WO2020071876A1 - High concentration trastuzumab with reduced viscosity or antigen-binding fragment stabilizing agent thereof - Google Patents

Abstract

The present invention relates to a stable pharmaceutical formulation comprising trastuzumab or an antigen-binding fragment thereof.

Description

High concentration trastuzumab with reduced viscosity or an antigen-binding fragment stabilizing agent thereof

The present invention relates to a stable pharmaceutical formulation comprising trastuzumab or an antigen-binding fragment thereof, and more particularly, to a formulation having a low viscosity and high stability and a method for manufacturing the same.

In order to improve patient convenience, the development of a technique for changing the administration method of an existing trastuzumab or antigen-binding fragment pharmaceutical product from intravenous injection to subcutaneous administration is underway. However, when administered subcutaneously, due to viscoelastic resistance in the subcutaneous tissue, the injectable volume is limited to 2.5 mL due to the back pressure and pain created during injection. To avoid this limitation, approaches have been used to enhance the absorption of injectables in the connective tissue by hydrolyzing the connective tissue in the subcutaneous tissue using a protein enzyme such as hyaluronidase. Medicines such as Herceptin ^® SC, MabThera ^® SC and RITUXAN HYCELA ^TM using this technology are currently sold or approved. However, when using a protein enzyme such as hyaluronidase, side effects such as allergic reactions and edema may occur. Therefore, there is still a need to develop a formulation having viscoelasticity suitable for subcutaneous injection without containing a protein enzyme.

High concentrations are required to produce a dosage form of trastuzumab or an antigen-binding fragment thereof that satisfies a dosage amount without degradation of subcutaneous tissue and has a limited volume of subcutaneous injection. However, highly concentrated trastuzumab or antigen-binding fragment formulations thereof tend to form highly viscous solutions due to the intermolecular interaction and macromolecular properties. Due to the increase in viscosity, trastuzumab or an antigen-binding fragment thereof is difficult to manufacture, transport, store and administer. In addition, due to the interaction between trastuzumab or an antigen-binding fragment thereof, problems such as aggregation or particle formation of trastuzumab or an antigen-binding fragment thereof may occur. Therefore, it is essential to develop a formulation capable of stabilizing the highly concentrated antibody trastuzumab or antigen-binding fragment thereof in various stress situations.

One aspect of the present invention provides a pharmaceutical formulation comprising a stable trastuzumab or antigen binding fragment thereof having low viscosity and high stability.

Another aspect of the invention provides a method for preparing the formulation.

One aspect of the present invention, (a) 100 mg / ml or more of trastuzumab or an antigen-binding fragment thereof; (b) buffering agents; (c) one or more selected from the group consisting of arginine, methionine and benzoate as stabilizers; And (d) provides a stable pharmaceutical formulation comprising a surfactant.

In another aspect of the present invention, a step of preparing a mixed solution by adding at least one selected from the group consisting of arginine, methionine and benzoate as a buffering agent and a stabilizing agent to a solvent, wherein at least 100 mg / ml tra is added to the mixed solution. Provided is a method for preparing a stable pharmaceutical preparation comprising adding stuuzumab or an antigen-binding fragment thereof, and adding a surfactant.

A stable pharmaceutical formulation according to an aspect of the present invention, a high concentration of trastuzumab or antigen binding thereof suitable for administration as an injection without containing a protein enzyme such as hyaluronidase, which may cause side effects in an individual after injection It was found to contain fragments at low viscosity and to improve the stability of trastuzumab or antigen binding fragments thereof. In addition, since the formulation is a stable pharmaceutical formulation comprising a high concentration of trastuzumab antibody or antigen-binding fragment thereof, it can be used not only for injection but also as various dosage forms.

1 is a graph showing the viscosity of the formulations of Examples 1 to 3 and Comparative Example 1.

2A, 2B, and 2C show the percent high molecular weight (% HMW),% monomer and percent low molecular weight substances in the formulations of Examples 1 and 2, and Comparative Example 1 under temperature stress conditions. This graph shows the results of analyzing the change in molecular weight (% LMW) by size-exclusion liquid chromatography (Size-Exclusion HPLC, SE-HPLC).

3A, 3B, and 3C are graphs showing the results of analyzing the charge change of the formulations of Example 1 and Comparative Example 1 with icIEF under temperature stress conditions.

Figure 4 is a graph showing the analysis of IgG changes in the formulations of Example 1 and Comparative Example 1 under temperature stress conditions by CE-SDS (NR).

5 is a graph showing the change in the HER2 binding rate of the formulations of Example 1 and Comparative Example 1 under temperature stress conditions.

[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 formulations of Examples and Comparative Examples

The formulations of Examples 1 to 3 and Comparative Example 1 were prepared.

First, with the composition of Table 1, trastuzumab (5 mM histidine buffer (pH 6.0), 60 mM trehalose, 0.04% polysorbate 20, 25 mg / mL trastuzumab, Samsung Bioepis Inc.) and buffering agent and stability excluding surfactant The topic was added to sterile distilled water in a 5L biotainer container made of polycarbonate to prepare a 5L buffer solution. 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 the cassette containing this trastuzumab solution was The dialysis was performed in a 2L beaker containing 1,600 mL of a buffer solution, and the 5 mM histidine buffer at pH 6.0 was exchanged with the buffer solution. Thereafter, trastuzumab was concentrated to 290 mg / mL. Concentration was achieved by passing the dialysis trastuzumab-containing solution through an Amicon Filter (Amicon ^™ Ultra 15 mL centrifugal filter, Merck) 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 shown in Table 1. The formulations of Examples 1 to 3 and Comparative Example 1 had a pH of about 5.5. Table 1 shows the composition of the formulations of Examples 1 to 3 and Comparative Example 1.

		Example 1	Example 2	Example 3	Comparative Example 1
Trastuzumab (mg / mL)		270	270	270	270
Buffering agent (mM)	Histidine	10	10	50	5
Stabilizer (mM)	Arginine	135	135	100	-
	Methionine	-	10		-
	Trehalose	-			60
	Sodium benzoate			100
Surfactant (w / v%)	Polysorbate 20	0.04	0.04	0.04	0.04

Other formulations of Examples 11 and 12 were prepared by the following method. Polycarbonate material of buffer and stabilizer except trastuzumab (5 mM histidine buffer (pH 6.0), 60 mM trehalose, 0.04% polysorbate 20, 25 mg / mL trastuzumab, Samsung Bioepis) and surfactant A 2L buffer solution was prepared by adding it to sterile distilled water in a 2L biotainer container. 400 mL of 40 mg / mL trastuzumab solution in 5 mM histidine buffer at pH 6.0 was placed in a labscale TFF system (Merck Millipore, USA), and then used Pellicon 3 Ultracel (Cat No. PXB100C50, Merck Millipore) (MWCO 50 kDa). Concentration proceeded to 150 mL. Thereafter, buffer exchange was performed using 1.8 mL, and then trastuzumab was concentrated to 290 mg / mL. Finally, to the concentrated trastuzumab solution, a surfactant prepared at a concentration of 20x, that is, 0.8 w / v%, in the prepared buffer solution was added to a concentration of 1x.

Test Example 1: Comparison of viscosity according to the combination of buffering agent and stabilizer

The viscosity of the prepared formulations of Examples 1 to 3 was measured to confirm that the formulation had a reduced viscosity despite the high concentration of trastuzumab.

Specifically, the viscosity of the formulation was measured using mVROC ^TM from Rheosense. 200 uL of the formulation was contained in a syringe of 250 uL, and viscosity was measured using a chip (C chip) with a sensor capable of measuring a range of 10 to 14,000 cP. In order to prevent interference between samples, the viscosity was measured using a sample of 150 uL remaining after each sample was flowed through 50 uL. All measurements were performed at 25 ° C. and measured at a shear rate between 100 and 10,000.

The results are shown in Table 2 and FIG. 1. The mean and standard deviation (SD) were calculated by performing 3 times for each formulation, and Table 2 shows the average per item for each formulation and the standard deviation in the lower row. It was confirmed that the viscosity of Examples 1 and 2 including the combination of histidine and arginine decreased by 91.6 cP and 81.7 cP, respectively, compared to Comparative Example 1. It was confirmed that Example 3 containing sodium benzoate had a lower viscosity than Comparative Example 1 and Examples 1 and 2.

Formulation	Actual protein concentration	Target pH	Viscosity (cP)	Osmotic pressure (mOsm / kg)
Example 1	271.3 (SD: 3.78)	5.5 ± 0.2	72.6 (SD: 7.8)	366 (SD: 52.2)
Example 2	271.2 (SD: 6.31)		62.7 (SD: 9.8)	373 (SD: 31.13)
Example 3	272.5 (SD: 5.0)		55.5 (SD: 4.1)	370 (SD: 0.4)
Comparative Example 1	275.8 (SD: 4.33)		154.3 (SD: 8.1)	216 mOsm / kg (SD: 49.1)

In Table 2, osmotic pressure was measured according to Test Example 7 below.

Test example  2: Benzoate  Viscosity change with concentration

As described for Example 3 of Test Example 1, benzoate reduced the viscosity of the formulation containing high concentration trastuzumab. Here, in the formulation containing high concentration of trastuzumab, the effect of benzoate on the viscosity change of the formulation containing high concentration of trastuzumab was confirmed by varying the concentration of benzoate.

Formulations comprising benzoate at different concentrations with high concentration trastuzumab contain 0.020 w / v% PS20 and have the compositions of Examples 4-7 in Table 3. The target pH of the formulations of Examples 4 to 7 is 5.5. The measured (N = 3) pH is Example 4: pH 5.59 (SD: 0.01), Example 5: pH 5.57 (SD: 0.02), Example 6: pH 5.59 (SD: 0.02), and Example 7: pH 5.55 (SD: 0.01).

The formulation of Example 4-7 was prepared in the same manner as in the preparation of the formulation of Example 3, except that the concentration of sodium benzoate and / or the composition of the stabilizer were varied.

The viscosity measurement results for the formulations of Examples 3 to 7 are shown in Table 3 below. The average and standard deviation were obtained by performing 3 times for each formulation, and the average for each formulation in Table 3 and the standard deviation are shown in the row below. However, in the case of Example 7, it was performed once per formulation. It was confirmed that benzoate had a viscosity significantly lower than that of Comparative Example 1, that is, 154.3 cP, in the entire range of 25 mM to 200 mM tested.

Formulation	Trastuzumab concentration (mg / mL)	Buffering agent	Stabilizer	Sodium benzoate	Viscosity (cP)	Osmotic pressure (mOsm / kg)
Example 4	270	50 mM His	100 mM Arg	25 mM	76.4 (SD: 2.3)	438 (SD: 5.4)
Example 5	270	50 mM His		50 mM	68.2 (SD: 5.1)	502 (SD: 7.8)
Example 6	270	50 mM His		75 mM	57.7 (SD: 1.6)	554 (SD: 9.3)
Example 3	270	50 mM His		100 mM	55.5 (SD: 4.1)	370 (SD: 0.4)
Example 7	270	50 mM His	-	200 mM	30.1	543.5

In Table 3, osmotic pressure was measured according to Test Example 7 below.

Test example  3: SE- HPLC  Stability comparison under stress conditions

It was confirmed by SE-HPLC whether the formulation of the above example can maintain stability while containing a high concentration of antibody under temperature or freeze-thaw stress conditions.

Specifically, 1 mL of each of the preparations of Examples 1 and 2 and Comparative Example 1 prepared above were placed in a 1.5 mL microtube (Axygen) made of polypropylene, and the tube was 40 ° C. in a stability thermostat (JEIO TECH). Was 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. As SE-HPLC, HPLC (Waters, USA; column: TSK-Gel G3000 SW _XL (Tosoh, Japan) was used. As a result, TSK-Gel G3000 SW _XL was connected, maintained at 25 ± 5 ° C., and 0.5 mL / min. The mobile phase was measured at the flow rate, and the temperature of the sample was maintained at 5 ± 3 ° C, and the injection amount was analyzed as 100 μg of protein The injection run time of the sample was 36 minutes The mobile phase was pH 6.8 and 100 mM sodium phosphate , And 200 mM shodium chloride-containing buffer was used and analyzed using absorbance at 280 nm The% HMW, and% monomer by SE-HPLC after storage for 4 weeks under temperature stress for the formulations of Examples 3 to 6. The same was carried out except for the measurement.

When the antibody in the formulation loses stability, aggregation increases the proportion of high molecular weight (HMW) substances, or the ratio of the monomer or low molecular weight (LMW) due to antibody degradation or disulfide bond destabilization. ) The proportion of the substance will be observed to decrease.

In addition, for freeze-thaw stress, 1 mL of the formulations of Examples 1 and 2 and Comparative Example 1 were respectively placed in a 15 mL microtube (Axygen) made of polypropylene, and

After placing the tube in a deep freezer (Forma ^TM , Thermo Scientific) maintained at -70 ± 10 ° C and maintaining it at 70 ° C for 18 hours to keep it frozen and frozen, the tube was taken out and at room temperature 1 It was left to thaw and kept in a thawed and thawed state. This process was repeated 5 times. When thawing, the container was gently inverted 5 or more times.

The results are shown in Tables 4 and 2 for Examples 1 and 2 and Comparative Example 1. The average and standard deviation were obtained by performing three times for each formulation, and the average for each formulation for Table 4 and the standard deviation are shown in the row below. It can be seen that Examples 1 and 2 have stability equal to or higher than that of Comparative Example 1 in temperature stress and freeze-thaw stress. The results for Examples 3 to 6 are shown in Table 5.

Formulation	Target pH			Starting point		40 ℃ / 4 weeks				F / T 5
		% HMW	% Monomer	% HMW	Δ% HMW	% Monomer	Δ% monomer	% HMW	Δ% HMW	% Monomer	Δ% monomer
Example 1	5.5 ± 0.2	1.06 (SD: 0.03)	98.35 (SD: 0.04)	2.99 (SD: 0.15)	+1.93 (SD: 0.16)	94.77 (SD: 0.29)	-3.58 (SD: 0.33)	1.15 (SD: 0.02)	+0.08 (SD: 0.06)	97.94 (SD: 0.06)	-0.41 (SD: 0.10)
Example 2		1.01 (SD: 0.02)	98.41 (SD: 0.03)	2.66 (SD: 0.02)	+1.65 (SD: 0.18)	95.11 (SD: 0.11)	-3.30 (SD: 0.07)	1.07 (SD: 0.03)	+0.06 (SD: 0.01)	98.00 (SD: 0.57)	-0.41 (SD: 0.08)
Comparative Example 1		2.39 (SD: 0.10)	97.06 (SD: 0.10)	5.75 (SD: 0.10)	+3.36 (SD: 0.41)	92.58 (SD: 0.25)	-4.48 (SD: 0.34)	2.58 (SD: 0.08)	+0.20 (SD: 0.03)	96.81 (SD: 0.09)	-0.25 (SD: 0.49)

Formulation	Target pH			Starting point		40 ℃ / 4 weeks
		% HMW	% Monomer	% HMW	Δ% HMW	% Monomer	Δ% monomer
Example 3	5.5 ± 0.2	0.94 (SD: 0.03)	98.99 (SD: 0.00)	4.72 (SD: 0.08)	3.78 (SD: 0.06)	92.67 (SD: 0.07)	-6.32 (SD: 0.04)
Example 4		0.89 (SD: 0.09)	98.82 (SD: 0.39)	5.48 (SD: 0.09)	4.59 (SD: 0.08)	91.68 (SD: 0.14)	-7.16 (SD: 0.51)
Example 5		0.88 (SD: 0.01)	99.06 (SD: 0.02)	6.97 (SD: 0.60)	6.10 (SD: 0.61)	90.33 (SD: 0.57)	-8.73 (SD: 0.58)
Example 6		0.87 (SD: 0.01)	0.98 (SD: 0.42)	12.43 (SD: 0.71)	11.57 (SD: 0.71)	84.92 (SD: 0.68)	-13.91 (SD: 0.70)

Test Example 4: Comparison of Stability at Temperature Stress Using icIEF It was confirmed by using icIEF (Imaging Capillary Isoelectric Focusing) that the formulation of the Example can maintain stability while containing a high concentration of antibody under temperature stress conditions.

Specifically, 1 mL of each of the preparations of Example 1 and Comparative Example 1 prepared above was placed in a 1.5 mL microtube (Axygen) made of polypropylene, and the tube was temperature of 40 ° C. in a stability thermostat (JEIO TECH). They were exposed to 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%. Charge variation value, that is,% acidification value, was measured using ICE3 (Protein Simple, USA) for the formulation stored for 4 weeks. The measurement sample is 2.9 uL of the formulation of Example 1 and Comparative Example 1, which is 1% (w / v) methylcellulose solution (101876, Protein Simple), pI marker 8.18 (Protein Simple, USA), 9.50 (Sigma, USA) , And a mixture of a suitable Pharmalyte (GE Healthcare) containing the pI section of trastuzumab was injected into the iCE3 system. The measurement results of charge variation values for the formulations of Example 1 and Comparative Example were processed using iCE CFR software.

The results are shown in Table 6 and FIG. 3. In the case of Example 1, the change in% acidity (Acidic) when temperature stress was applied at 40 ° C for 4 weeks was lower than that of Comparative Example 1. This shows that the formulation of Example 1 has a lower charge variation than Comparative Example 1. Therefore, it can be seen that Example 1 has a more constant charge than Comparative Example 1 at temperature stress.

Formulation	Temperature stress (40 ℃): icIEF
	Starting point			2 weeks			4 weeks
	%acid	%neutrality	% Basic	%acid	%neutrality	% Basic	%acid	%neutrality	% Basic
Example 1	39.50	52.13	8.37	50.35	41.41	8.24	58.50	33.60	7.90
	SD: 0.29	SD: 0.53	SD: 0.33	SD: 0.43	SD: 0.83	SD: 0.43	SD: 0.64	SD: 0.91	SD: 0.28
Comparative Example 1	41.34	50.48	8.18	62.47	31.84	5.69	74.87	20.16	4.97
	SD: 0.44	SD: 0.32	SD: 0.48	SD: 0.53	SD: 0.37	SD: 0.22	SD: 0.12	SD: 0.25	SD: 0.26

In Table 6,% acidity,% neutrality, and% basicity represent charge variation values of proteins. Measuring these is a test method to identify the charge variant of the protein. Each protein has a different amino acid sequence and different formulations, so there may be charge variation. When the protein changes due to stress, aggregation or charge changes. Therefore, the charge variation of the protein can be a factor that confirms its stability.

Test example  5: CE-SDS ( NR Stability comparison at temperature stress using)

It was confirmed by using a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) that the formulation of the above example can maintain stability while containing a high concentration of the antibody under temperature stress conditions.

Specifically, 1 mL of each of the preparations of Example 1 and Comparative Example 1 prepared above was placed in a 1.5 mL microtube (Axygen) made of polypropylene, and the tube was temperature of 40 ° C. in a stability thermostat (JEIO TECH). They were exposed to 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%. IgG, 2H1L, and other proteins were measured by CE-SDS for preparations stored for 4 weeks. After 4 weeks,% 2H1L (2 heavy and 1 light chain peptides),% other peptides and% IgG were evaluated based on the weight of the antibody tested.

CE-SDS conditions were non-reduced (NR) conditions. Specifically, the sample was pretreated by mixing the measurement sample, SDS-MW sample buffer, internal standard, and iodo acetamide solution and heating for 5 minutes in a 70 ± 2 ° C heating block. 90 μL of the pre-treated sample was placed in a PCR tube. The PCR tube was placed in a universal vial containing a running buffer and loaded into a PA800 plus system. The cartridge temperature and sample temperature of the instrument were maintained at 25 ° C and 15 ° C, respectively. Relative migration time (RMT, relative migration time) of the analytical sample is obtained from the obtained electrophoresis as the peak migration time (analyte) of the analysis sample to the peak migration time (10k Da internal standard) of the internal standard. It can be divided. In addition, the CPA (Corrected peak area) can be calculated by dividing the peak migration time by the peak area, and% CPA can be calculated by dividing each CPA by the sum of the CPAs of all peaks.

The results are shown in Table 7 and FIG. 4. Compared to Comparative Example 1, the Δ% IgG value of Example 1 was confirmed to be an equivalent level. Therefore, it was confirmed that Example 1 has stability equal to or higher than that of Comparative Example 1 in temperature stress. In Table 7, Δ% IgG is the% IgG value at week 4 minus the% IgG value at the starting point.

Formulation	Temperature stress (40 ℃): CE-SDS (NR)
	Starting point			2 weeks			4 weeks
	% 2H1L	% Other Protein	% IgG	% 2H1L	% Other Protein	% IgG	% 2H1L	% Other Protein	% IgG	% ΔIgG
Example 1	0.52	2.59	96.68	2.78	2.38	94.20	2.03	2.69	94.45	-2.23
	SD: 0.17	SD: 0.65	SD: 0.81	SD: 0.07	SD: 0.11	SD: 0.18	SD: 0.02	SD: 0.04	SD: 0.14	SD: 0.85
Comparative Example 1	0.41	2.21	96.70	2.16	1.82	93.89	1.85	2.47	93.71	-2.98
	SD: 0.03	SD: 0.06	SD: 0.10	SD: 0.28	SD: 0.16	SD: 0.35	SD: 0.03	SD: 0.03	SD: 0.12	SD: 0.20

Test Example 6: Comparison of maintenance of activity at temperature stress It was confirmed that the formulation of the above example maintains stability under temperature stress conditions and consequently maintains activity.

Specifically, the preparation of Example 1 and Comparative Example 1 prepared above was evaluated by measuring the binding ratio to HER2 to maintain activity. The HER2 binding rate was measured by a binding assay based on fluorescence resonance energy transfer.

Reference standards, assay controls and samples were diluted to a concentration of 0.0488-100 μg / mL and placed in each well of a 96-well plate. The concentration of trastuzumab was measured using Nanodrop (Thermo, USA). Then, after adding traustumab (PerkinElmer, USA) labeled with europium and HER2 labeled with Cy5, the light was blocked against a 96-well plate and stirred at 25 ± 1 ° C. at 500 rpm for 60 minutes. Incubated. Then, the sample contained in each well was excited at 340 nm to measure the fluorescence energy emitted at 665 nm with an Envision multimode plate reader (Perkin Elmer, USA). Each% relative binding activity (RBA) value was obtained.

The results are shown in Table 8 below and FIG. 5. Compared to Comparative Example 1, it was confirmed that the binding ratio of the formulation of Example 1 to HER2 was equal or higher. Therefore, it was confirmed that Example 1 has stability equal to or higher than that of Comparative Example 1 in temperature stress, and the effect of maintaining the activity of the antibody is also improved.

Formulation	Temperature stress (40 ℃): bonding
	Starting point	2 weeks	4 weeks
% RBA	% RBA	% RBA
Example 1	97.00	94.00	85.00
	SD: 2.08	SD: 8.72	SD: 6.00
Comparative Example 1	98.00	93.00	68.33
	SD: 2.89	SD: 3.61	SD: 3.06

Test Example 7: Confirmation of Characteristics of Formulation Containing High Concentration of Trastuzumab Even when the concentration of trastuzumab contained in the formulation was increased, it was confirmed whether the formulation of the present invention was capable of maintaining the characteristics suitable for subcutaneous or intravenous administration. If a high concentration of trastuzumab is included and the osmotic pressure and viscosity are excessively increased, the formulation may not be suitable for subcutaneous or intravenous administration.

Specifically, Examples 8 to 12 were prepared with the composition of Table 8 by varying only the concentration of trastuzumab based on Example 1. Trastuzumab used trastuzumab as in Preparation Example. The viscosity was measured in the same manner as in Test Example 1. Table 9 shows the results. Although Examples 1 and 8 to 12 both contained high concentrations of 200 mg / mL or higher antibody, it was confirmed that they had a low viscosity in all the Examples tested.

For Examples 11 and 12, osmotic pressure was further measured. Osmolarity can be measured, for example, by a freezing point method with an Advanced osmometer 2020 (Fisher scientific, USA). Specifically, a sample of 20 μL of a high-concentration formulation diluted 1/2 with a buffer solution was measured twice by using a freezing point method. In the case of a high concentration formulation, it may not be directly measured due to an increase in viscosity and a change in the freezing point. In this case, the osmotic pressure is indirectly calculated using the calculation formula of "high concentration formulation osmotic pressure = {[(high concentration formulation osmotic pressure diluted 1/2 with buffer solution-osmotic pressure of buffer solution) * 2] + buffer osmotic pressure" using the used buffer solution. I can get it. Osmolarity concentrations of the formulations of the present invention may be, for example, 200 to 400 mOsm / kg, 250 to 380 mOsm / kg, or 270 to 360 mmol / kg. As a result, as shown in Table 9, Examples 11 and 12 were also impossible to contain high concentration antibodies of 200 mg / mL or more and had low viscosity, and the osmolality was measured to have 355 mOsm / kg and 320 mOsm / kg.

Thus, the formulations of the present invention contain a high concentration of trastuzumab or antigen binding fragments of 200 mg / mL or more, but have viscosity and osmotic pressure suitable for subcutaneous or intravenous administration.

Formulation	Trastuzumab concentration (mg / mL)	Target pH	Buffering agent	Stabilizer	Surfactants	Measured trastuzumab concentration (mg / mL)	Viscosity (cP)
Example 1	270	5.5 ± 0.2	10 mM His	135 mM Arg	0.04% PS 20	264.7 (N = 3, SD: 1.06)	68.1 (N = 3, SD: 3.39)
Example 8	250		10 mM His	135 mM Arg	0.04% PS 20	254.5 (N = 3, SD: 1.15)	52.3 (N = 3, SD: 1.02)
Example 9	240		10 mM His	135 mM Arg	0.04% PS 20	241.0 (N = 3, SD: 2.00)	36.1 (N = 3, SD: 3.85)
Example 10	220		10 mM His	135 mM Arg	0.04% PS 20	221.8 (N = 3, SD: 2.35)	22.7 (N = 3, 0.04)
Example 11	270		10 mM His	135 mM Arg20 mM Met	0.04% PS 20	267.9 (N = 3, SD: 7.2)	73.0 (N = 3, SD: 5.6)
Example 12	240		10 mM His	135 mM Arg20 mM Met	0.04% PS 20	248.8 (N = 3, SD: 2.8)	34.5 (N = 3, SD: 0.3)

Test Example 8: Confirmation of the Stability of the Formulation Containing High Concentration of Trastuzumab or an Antigen-binding Fragment thereof Measured using HPLC, and icIEF, and evaluated through HER2 binding level.

In the test, stability was evaluated after applying stress to Examples 3 to 7, and Examples 11 and 12 in the same manner as in Test Examples 3 to 6 above. Stability was evaluated under temperature stress conditions for 4 weeks for Examples 3 to 6 and 2 weeks for Example 7. The results are shown in Tables 10 to 12. However, the temperature stress was evaluated for 2 weeks at a high temperature of 40 ° C and a low temperature of 5 ° C. Resistance to shear stress was performed by stirring the container containing the sample with an orbital shaker at 400 rpm for 72 hours and measuring using SE-HPLC and icIEF. Despite the high concentration of trastuzumab, it can be seen that it has excellent stability in all evaluation items.

Formulation	Target pH	Formulation	SE-HPLC				icIEF
			% HMW (start point)	% HMW (F / T 5)	% HMW (0 rpm, 72 h)	% HMW (400 rpm, 72 h)	% Acidity (starting point)	% Acidity (F / T 5)	% Acidity (0 rpm, 72 h)	% Acidity (400 rpm, 72 h)
Example 11	5.5 ± 0.2	10 mM His, 135 mM Arg, 20 mM Met0.04% PS 20	1.00 [SD: 0.03]	1.06 [SD: 0.04]	1.18 [SD: 0.05]	1.22 [SD: 0.04]	39.17 [SD: 0.29]	39.86 [SD: 0.24]	39.84 [SD: 0.50]	39.78 [SD: 0.50]
Example 12			0.93 [SD: 0.04]	1.02 [SD: 0.02]	1.11 [SD: 0.04]	1.13 [SD: 0.05]	39.11 [SD: 0.71]	39.95 [SD: 0.39]	40.52 [SD: 0.62]	41.03 [SD: 0.33]

Formulation	Temperature stress (40 ℃): CE-SDS (NR)
	Starting point			2 weeks			4 weeks
	% 2H1L	% Other Protein	% IgG	% 2H1L	% Other Protein	% IgG	% 2H1L	% Other Protein	% IgG	% ΔIgG
Example 11	1.44	1.33	97.23	1.86	2.58	95.57	2.93	4.20	92.86	-4.36
	SD: 0.05	SD: 0.04	SD: 0.09	SD: 0.32	SD: 0.19	SD: 0.28	SD: 0.13	SD: 0.17	SD: 0.08	SD: 0.14
Example 12	1.54	1.28	97.17	1.93	2.34	95.73	2.35	3.72	93.50	-3.24
	SD: 0.01	SD: 0.06	SD: 0.07	SD: 0.32	SD: 0.04	SD: 0.28	SD: 0.22	SD: 0.09	SD: 0.55	SD: 0.24

Formulation	Target pH	Formulation	HER2 binding
			% RBA (start point)	% RBA (5 ℃ / 2 Wk)	% RBA (40 ℃ / 2 Wk)
Example 11	5.5 ± 0.2	10 mM His, 135 mM Arg, 20 mM Met0.04% PS 20	99.3 [SD: 14.5]	94.0 [SD: 5.3]	92.3 [SD: 9.1]
Example 12			93.0 [SD: 10.4]	93.3 [SD: 1.2]	83.7 [SD: 1.5]

Test Example 9: Comparison of stability according to the concentration of the buffering agent and the stabilizer It was confirmed the stabilizing effect of the high concentration of trastuzumab or an antigen-binding fragment thereof at various combinations and concentrations of the buffering agent and the stabilizer. Stabilization was evaluated by measuring using temperature- or freeze-thaw stress conditions and then using SE-HPLC and icIEF.

The combinations and concentrations of the tested buffers and stabilizers are shown in Table 13, and were measured using SE-HPLC after applying stress as in Test Examples 3 and 4. In addition, the viscosity was measured in the same manner as described in Test Example 1. As a result, the viscosity at the start of Examples 20 to 27 was 66.6, 70.2, 81.1, 73.9, 68.7, 66.3, 80.5, and 72.2 cP, respectively. Examples 20-27 included trastuzumab and PS20, their concentrations were 270 mg / mL and 0.04 w / v%, respectively. Table 13 shows the results.

Formulation	Target pH	His (mM)	Arg (mM)	Δ% HMW40 ℃ / 4 wk	Δ% monomer 40 ℃ / 4 wk	Δ% acidity 40 ℃ / 4 wk	Δ% neutral 40 ℃ / 4 wk	Δ% HMWF / T
Example 20	5.0	20	100	2.43	-3.02	+1.65	-29.13	+0.02
Example 21	5.0	20	200	2.79	-2.08	-2.67	-28.38	-0.03
Example 22	6.0	0	100	2.57	-3.12	+12.35	-30.32	-0.03
Example 23	6.0	20	100	2.20	-2.62	+23.96	-33.75	-0.05
Example 24	6.0	0	200	1.90	-2.36	+12.78	-30.20	-0.04
Example 25	6.0	20	200	1.81	-2.32	+24.00	-34.55	-0.10
Example 26	5.5	10	150	2.13 [N = 3, SD: 0.07	-2.66 [N = 3, SD: 0.05]	+9.93 [N = 3, SD: 5.15]	-29.49 [N = 3, SD: 1.20]	-0.05 [N = 3, SD: 0.07]
Example 27	5.5	20	200	1.80 [N = 3, SD: 0.13]	-2.45 [N = 3, SD: 0.11]	5.27 [N = 3, SD: 2.79]	-29.33 [N = 3, SD:	-0.03 [N = 3, SD: 0.01]

As shown in Table 13, stability of the composition according to changes in pH, histidine and arginine concentrations was confirmed.

Test Example 10: Comparison of stability and characteristics of formulations containing benzoate

By varying the concentration of benzoate in various buffering and stabilizing agent combinations and concentrations, the stabilizing effect and viscosity and osmotic characteristics of the high concentration trastuzumab were confirmed. Stabilization was evaluated by measuring using temperature- or freeze-thaw stress conditions and then using SE-HPLC and icIEF.

The tested buffering agents, stabilizers and benzoate concentrations are as shown in Tables 14 to 17, and were measured using SE-HPLC and icIEF after applying stress as in Test Examples 3 and 4. In addition, viscosity and osmotic pressure were measured as in Test Example 7. The formulations of Examples 28-31 included trastuzumab and PS20, the concentrations of which were 270 mg / mL and 0.04 w / v%, respectively. Examples 28 to 31 were adjusted to pH at pH 6.0 to 7.0. In addition, because there may be errors in manufacturing and measurement, the target pH ± 0.2 was set as acceptance criteria. The median value is 6.5.

The results are shown in Tables 14 to 17. Table 14 shows the viscosity and osmotic pressure measured at the time point before exposure to the temperature stress condition of 40 ℃ for 4 weeks. Table 15 shows the results measured using SE-HPLC at temperature stress. Table 16 shows the results measured using icIEF at temperature stress. Table 18 shows the results measured using SE-HPLC in freeze-thaw stress.

Formulation	His (mM)	Arg (mM)	Sodium benzoate (mM)	Met (mM)	Measured pH	Measured trastuzumab concentration (mg / mL)	Osmotic pressure (mOsm / kg)	Viscosity (cP)
Example 28	10.0	30.0	75.0	20.0	6.02	277.5	336	72.1
Example 29	50.0	30.0	75.0	0.0	6.01	277.9	372	71.3
Example 30	10.0	100.0	75.0	0.0	5.95	275.5	410	76.8
Example 31	50.0	100.0	75.0	20.0	5.98	269.7	493	60.8

As shown in Table 14, Examples 28 to 31 show the osmotic pressure and viscosity change according to the concentration change of pH, histidine, arginine, sodium benzoate, and methionine.

Formulation	His (mM)	Arg (mM)	Sodium benzoate (mM)	Met (mM)	% HMW (start point)	% Monomer (start point)	% LMW (start point)	% HMW (40 ℃ / 4 wk)	% Monomer (40 ℃ / 4 wk)	% LMW (40 ℃ / 4 wk)	Δ% HMW (40 ℃ / 4 wk)
Example 28	10.0	30.0	75.0	20.0	1.21	98.11	0.68	4.12	93.67	2.21	2.91
Example 29	50.0	30.0	75.0	0.0	1.10	98.25	0.65	4.31	93.3	2.39	3.21
Example 30	10.0	100.0	75.0	0.0	1.21	98.15	0.65	4.23	93.42	2.35	3.02
Example 31	50.0	100.0	75.0	20.0	0.96	98.38	0.66	3.26	94.39	2.35	2.30

As shown in Table 15, Examples 30-51 show changes in molecular size under temperature stress with varying concentrations of pH, histidine, arginine, sodium benzoate, and methionine.

Formulation	His (mM)	Arg (mM)	Sodium benzoate (mM)	Met (mM)	% Acidity (starting point)	% Neutral (start point)	% Basic (start point)	% Acid (40 ℃ / 4 wk)	% Neutral (40 ℃ / 4 wk)	% Basic (40 ℃ / 4 wk)	Δ% acidity (40 ℃ / 4 wk)
Example 28	10.0	30.0	75.0	20.0	33.03	53.26	13.71	52.37	22.23	25.39	19.34
Example 29	50.0	30.0	75.0	0.0	33.17	52.86	13.98	52.37	21.51	26.12	19.20
Example 30	10.0	100.0	75.0	0.0	32.19	53.24	14.57	50.46	22.60	26.94	18.27
Example 31	50.0	100.0	75.0	20.0	32.87	53.52	13.61	53.87	21.25	24.88	21.00

As shown in Table 16, Examples 30 to 51 show charge change under temperature stress according to changes in concentration of pH, histidine, arginine, sodium benzoate, and methionine.

Formulation	His (mM)	Arg (mM)	Sodium benzoate (mM)	Met (mM)	% HMW (start point)	% Monomer (start point)	% LMW (start point)	% HMW (F / T 5)	% Monomer (F / T 5)	% LMW (F / T 5)	Δ% HMW (F / T 5)
Example 28	10.0	30.0	75.0	20.0	1.21	98.11	0.68	1.30	97.86	0.85	0.09
Example 29	50.0	30.0	75.0	0.0	1.10	98.25	0.65	1.18	97.97	0.85	0.08
Example 30	10.0	100.0	75.0	0.0	1.21	98.15	0.65	1.27	97.86	0.86	0.06
Example 31	50.0	100.0	75.0	20.0	0.96	98.38	0.66	1.02	98.14	0.84	0.06

As shown in Table 17, Examples 30-51 show changes in molecular size under freeze-thaw stress with varying concentrations of pH, histidine, arginine, sodium benzoate, and methionine.

All technical terms used in the present invention, unless defined otherwise, are used in a sense as generally understood by a person 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 present invention, (a) 100 mg / ml or more of trastuzumab or an antigen-binding fragment thereof; (b) buffering agents; (c) one or more selected from the group consisting of arginine, methionine and benzoate as stabilizers; And (d) provides a stable pharmaceutical formulation comprising a surfactant.

The pharmaceutical formulation may be liquid, or solid. The pharmaceutical preparation may be in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, aerosol, ex, injection, transdermal or suppository. The solid may be a dry one of the liquid.

The formulation of the present invention includes a high concentration of trastuzumab or an antigen-binding fragment thereof, but can maintain low viscosity and stability of trastuzumab or an antigen-binding fragment thereof.

The term “antibody” refers to a target molecule, ie, an immunoglobulin molecule consisting of a polypeptide chain capable of binding an antigen. Such antibodies can be polyclonal antibodies, monoclonal antibodies, recombinant antibodies, single chain antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibodies with polyepitope specificity, or multispecific antibodies (e.g., dia Body). The term “antigen-binding fragment” is a fragment capable of binding to an antigen in an antibody, including, but not limited to, Fab, F (ab ′) ₂ or Fv, for example. In one embodiment, the antibody can be a monoclonal antibody. 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 the desired HER2 receptor. 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.

The concentration of trastuzumab or an antigen-binding fragment thereof included in the formulation of the present invention may be 100 mg / ml or more. For example, the concentration may be 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mg / ml or more. 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. The concentration of the trastuzumab antibody or antigen-binding fragment thereof is, for example, 100-300 mg / mL, 110-300 mg / ml, 120-300 mg / ml, 130-300 mg in one embodiment (Group 1) / ml, 140-300 mg / ml, 150-300 mg / ml, 160-300 mg / ml, 170-300 mg / ml, 180-300 mg / ml, 190-300 mg / ml, 200-300 mg / mL, 210-300 mg / ml, 220-300 mg / mL, 110-290 mg / ml, 120-290 mg / ml, 130-290 mg / ml, 140-290 mg / ml, 150-290 mg / mL , 160 to 290 mg / ml, 170 to 290 mg / ml, 180 to 290 mg / ml, 190 to 290 mg / ml, 200 to 290 mg / mL, 210 to 290 mg / ml, 220 to 290 mg / mL, 110 to 280 mg / ml, 120 to 280 mg / ml, 130 to 280 mg / ml, 140 to 280 mg / ml, 150 to 280 mg / mL, 160 to 280 mg / ml, 170 to 280 mg / ml, 180 To 280 mg / ml, 190 to 280 mg / ml, 200 to 280 mg / mL, 210 to 280 mg / ml, 220 to 280 mg / mL, 110 to 270 mg / ml, 120 to 270 mg / ml, 130 to 270 mg / ml, 140-270 mg / ml, 150-270 mg / mL, 160-270 mg / ml, 170-270 mg / ml, 180-270 mg / ml, 190-270 mg / ml, 200-270 mg / mL, 210-270 mg / ml, 220-270 mg / mL, in another embodiment (Group 2) 230-300 mg / ml, 240-300 mg / mL, 230-290 mg / ml, 240- 290 mg / mL, 230-280 mg / ml, 240-280 mg / mL, 230-270 mg / ml, 240-270 mg / mL, in another embodiment (Groups 3,4,5) 250 To 300 mg / mL, 260 to 300 mg / mL, 270 to 300 mg / mL, 250 to 290 mg / mL, 260 to 290 mg / mL, 265 to 290 mg / mL, 250 to 280 mg / mL, 260 to 280 mg / mL, 265 to 280 mg / mL, 250 to 275 mg / mL, 260 to 275 mg / mL, and 265 to 275 mg / mL. The concentration of trastuzumab or antigen-binding fragment thereof can be freely adjusted within a range that does not substantially affect the stability, desired viscosity and pH of the formulation of the present invention. The concentration of trastuzumab or antigen-binding fragment thereof may contribute to its stability in the formulation, viscosity of the formulation suitable for administration, and maintenance of the pH of the formulation.

The formulations 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 or a salt thereof, but is not limited thereto. In one embodiment, the buffering agent can be histidine

In one embodiment, the concentration of histidine may be 1 to 150 mM, specifically, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or more. The concentration of the histidine in one embodiment (Group 2,5) 1 to 100 mM, 1 to 90 mM, 1 to 80 mM, 1 to 70 mM, 1 to 60 mM, 1 to 55 mM, 5 to 100 mM, 5 to 90 mM 5 to 80 mM, 5 to 70 mM, 5 to 60 mM, 5 to 55 mM, 7 to 100 mM, 7 to 90 mM, 7 to 80 mM, 7 to 70 mM, 7 to 60 mM, 7 To 55 mM, 10 to 100 mM, 10 to 90 mM, 10 to 80 mM, 10 to 70 mM, 10 to 60 mM, 10 to 55 mM, 10 to 50 mM, in other embodiments (Group 1) 1 to 40 mM, 1 to 30 mM, 1 to 25 mM, 5 to 40 mM, 5 to 30 mM, 5 to 25 mM, 10 to 20 mM, in another embodiment (Group 4) 40mM to 60mM , 45mM to 60mM, 45mM to 55mM and in another embodiment (Group 3) may be 1 to 20mM, 1 to 15mM, 5 to 20mM, 5 to 15mM. The concentration of histidine can be freely adjusted within a range that does not affect the desired pH of the formulation of the present invention and does not affect the stability of trastuzumab or an antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof. have. The histidine may be used as a buffering agent. The histidine may be histidine HCl.

One aspect of the present invention may include a stabilizer. The stabilizer may be at least one selected from the group consisting of metal salts, amino acids and benzoates, but is not limited thereto. The stabilizer may be one that does not contain sugar, sugar alcohol, sugar acid, polyol, or a combination thereof. The sugar, sugar alcohol or sugar acid is glucose, fructose, galactose, sucrose, lactose, maltose, trehalose, fructooligosaccharide, galactoligosaccharide, metoligosaccharide, starch, glycogen, cellulose, chitin, pectin, glycerol, erythritol, Threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, bolitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol , Aldonic acid, ulonic acid, uronic acid, aldaric acid, stachyose, sorbose, xylose, ribose, myoinicitose, myoinisitol, and polyethylene glycol.

In one embodiment, the stabilizer may be selected from one or more of arginine, methionine, or benzoate. In this case, the formulation may not contain other stabilizers. For example, the formulation may not include sugar, sugar alcohol, sugar acid, or a combination thereof as a stabilizer. Specifically, the formulation may not include trehalose.

Arginine as used herein includes arginine monohydrochloride. Arginine monohydrochloride may exist in L-form or D-form, or a mixture of the two. It is also known as (2S) -2-amino-5-[(aminoiminomethyl) amino] pentanoic acid monohydrochloride, arginine hydrochloride, and arginine · HCl. Arginine monohydrochloride can be prepared by neutralizing arginine with hydrochloric acid. The concentration of arginine may be 1 to 300 mM. The concentration of the arginine in one embodiment (Group 3, 5) 1 to 100 mM, 5 to 100 mM, 10 to 100 mM, 15 to 100 mM, 20 to 100 mM, 25 to 100 mM, 30 to 100 mM day In another embodiment (Group 1) 100-300mM, 100-290mM, 100-280mM, 100-270mM, 100-260mM, 100-250mM, 100-240mM, 100-230mM, 100-220mM, 100-210mM , 100 to 200mM, in another embodiment (Group 2) 30 to 300mM, 50 to 300mM, 60 to 300mM, 70 to 300mM, 80 to 300mM, 90 to 300mM, 100 to 300mM, 110 to 300mM, 120 300 to 300 mM, 130 to 300 mM, 30 to 250 mM, 50 to 250 mM, 60 to 250 mM, 70 to 250 mM, 80 to 250 mM, 90 to 250 mM, 100 to 250 mM, 110 to 250 mM, 120 to 250 mM, 130 to 250 mM, 30 to 200 mM , 50 to 200mM, 60 to 200mM, 70 to 200mM, 80 to 200mM, 90 to 200mM, 100 to 200mM, 110 to 200mM, 120 to 200mM, 130 200mM, it may be a 30 to 150mM, 50 to 150mM, 60 to 150mM, 70 to 150mM, 80 to 150mM, 90 to 150mM, 100 to 150mM, 110 to 150mM, 120 to 150mM, 130 to 150mM, 130 to 145mM. The concentration of arginine can be freely adjusted within a range that does not affect the desired pH of the formulation of the present invention and does not affect the stability of trastuzumab or antigen-binding fragments thereof. The arginine may be used as a stabilizer for increasing the stability of trastuzumab or an antigen-binding fragment thereof.

The concentration of benzoate may be 10 mM or more. The concentration of the benzoate in one embodiment (Group 3) 10 to 200 mM, 15 to 200 mM, 20 to 200 mM, 10 to 190 mM, 15 to 190 mM, 20 to 190 mM, 10 to 180 mM, 15 to 180 mM , 20 to 180 mM, 10 to 170 mM, 15 to 170 mM, 20 to 170 mM, 10 to 160 mM, 15 to 160 mM, 20 to 160 mM, in another embodiment (Group 5) 30 to 200 mM, 40-200 mM, 50-200 mM, 60-200 mM, 70-200 mM, 30-190 mM, 40-190 mM, 50-190 mM, 60-190 mM, 70-190 mM, 30-180 mM, 40- 180 mM, 50-180 mM, 60-180 mM, 70-180 mM, 30-170 mM, 40-170 mM, 50-170 mM, 60-170 mM, 70-170 mM, 30-160 mM, 40-160 mM, 50 to 160 mM, 60 to 160 mM, 70 to 160 mM, and in other embodiments (Group 4) 150 to 220 mM, 150 to 215 mM, 150 to 210 mM, 160 to 220 mM, 160 to 215 mM, 160 to 210 mM, 170 to 220 mM, 170 to 215 mM, 170 to 210 mM, 180 to 220 mM, 180 to 215 mM, 180 to 210 mM, 190 to 220 mM, 190 to 215 mM, 190 to 210 mM, 195 to 205 mM, . The concentration of benzoate can be freely adjusted within a range that does not affect the stability of trastuzumab or an antigen-binding fragment thereof.

The benzoate may be prepared by reacting benzoic acid with a metal salt such as NaCl, KCl, NaF, KBr, NaBr, Na ₂ SO ₄ , NaSCN, or K ₂ SO ₄ , but is not limited thereto. In one embodiment, the benzoate may be sodium benzoate. The benzoate can be freely selected within a range that does not affect the desired pH of the formulation of the invention and does not affect the stability of trastuzumab or antigen-binding fragments thereof. The benzoate may be used as a stabilizer for increasing the stability of trastuzumab or an antigen-binding fragment thereof.

The concentration of the methionine may be 1 to 50 mM, in one embodiment, 1 to 40 mM, 1 to 30 mM, 1 to 25 mM, 1 to 20 mM, 5 to 40 mM, 5 to 30 mM, 5 to 20 mM, 8 to 40 mM, 8 to 30 mM, 8 to 20 mM, 10 to 40 mM, 10 to 30 mM. The methionine may be used as a stabilizer for increasing the stability of trastuzumab or an antigen-binding fragment thereof.

In one embodiment, the formulation may not include hyaluronidase. Hyaluronidase includes all of a variety of commonly known hyaluronidases or modified hyaluronidases, such as the enzyme protein having the amino acid sequence of GenBank: AAC70915.1 in humans. The hyaluronidase enzyme may be a glycoprotein, for example, rHuPH20.

The formulation of the present invention does not contain hyaluronidase, but has a low viscosity. The viscosity of the formulation may be 100 cP or less, for example, 95 cP or 90 cP or less. In one embodiment, the viscosity of the formulation is 1 to 100 cP, 22 to 100 cP, 22 to 86 cP, 20 to 88 cP, 17 to 91 cP, 12 to 96 cP, 22 to 74 cP, 20 to 76 cP, 17 to 79 cP, 12 to 84 cP, 53 to 82 cP, 51 to 84 cP, 48 to 87 cP, 43 to 92 cP, about 30 cP, 28 to 32 cP, 25 to 35 cP, 20 to 40 cP, 34 To 73 cP, 32 to 75 cP, 29 to 78 cP, 24 to 83 cP, 47 to 86 cP, 45 to 88 cP, 42 to 91 cP, or 37 to 96 cP.

In one embodiment, the surfactant is a 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), and specifically, a nonionic surfactant.

The nonionic surfactant is, for example, polysorbate, that is, polyoxyethylene sorbitan fatty acid ester or a derivative thereof, poloxamer, that is, polyoxyethylene-polyoxypropylene copolymer or a derivative thereof, and sorbitan ester. It may be one or more selected from the group consisting of. The polysorbate can be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a combination thereof. The poloxamer may be poloxamer 188.

The surfactant has a concentration of 0.01 to 0.7w / v%, for example, 0.01 to 0.1w / v%, 0.01 to 0.09 w / v%, 0.01 to 0.08 w / v%, 0.01 to 0.07 w / v%, 0.02 to 0.06 w / v%, or 0.03 to 0.05 w / v%.

In one embodiment, the pH of the formulation may be 4.0 to 7.5. In other embodiments, the pH of the formulation is 5.0 to 7.0, 4.9 to 7.1, 4.8 to 7.2, 4.7 to 7.3, 4.5 to 7.5, 5.3 to 7.0, 5.2 to 7.1, 5.1 to 7.2, 5.0 to 7.3, 4.8 to 7.5, About 5.5, 5.4 to 5.6, 5.3 to 5.7, 5.2 to 5.8, 5.1 to 5.9, 5.0 to 6.0, 4.8 to 6.2, 5.5 to 6.5, 6.0 to 7.0, 5.9 to 7.1, 5.8 to 7.2, 5.7 to 7.3, or 5.5 to May be 7.5. The pH of the formulation can be freely adjusted within a range suitable for preventing aggregation and maintaining activity of trastuzumab or an antigen-binding fragment thereof.

Stabilizers, buffering agents, and surfactants as defined above may mean different things.

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.

The trastuzumab or antigen-binding fragment thereof exhibits any signs of aggregation, precipitation and / or denaturation upon visual inspection for color and / or transparency or as measured by ultraviolet (UV) light scattering or size exclusion chromatography. If not visible, it retains physical stability within the pharmaceutical formulation.

“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. 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 10% or less, 5% or less, or 2.5% or less relative to the initial value.

The antibody or antigen-binding fragment thereof retains the biological activity in the pharmaceutical agent when the antibody or antigen-binding fragment thereof is biologically active for its intended purpose in the pharmaceutical preparation. The biological activity is, for example, about 30%, about 20%, about 10%, about 5%, about 3 of the biological activity of the antibody or antigen-binding fragment thereof in the pharmaceutical agent at the time of manufacture of the pharmaceutical agent. % Or retain biological activity if within analytical error. The biological activity can be determined, for example, in antigen binding assays.

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 the chemically modified form of trastuzumab. 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 an enzyme-linked immunosorbent assay (ELISA). ELISA analysis measures the ratio (%) of trastuzumab binding to the HER2 receptor. ELISA can be performed using a HER2 ELISA kit (R & D system). After reacting with the trastuzumab sample and the HER2 receptor on the ELISA substrate, the degree of binding can be determined by measuring absorbance at 450 nm to obtain a relative binding rate (%).

Stabilization may include temperature stress, for example, 1 to 4 weeks or 8 weeks at 40 ° C; Freeze-thaw stress, eg, -70 ° C freezing and thawing at room temperature, 5 repetitions; Alternatively, it can be evaluated by measuring stirring stress, for example, by applying 400 rpm rotational force in a centrifuge for 72 hours, and then measuring% HMW,% monomer and / or% LMW using SE-HPLC. In one embodiment, the formulations of the invention may have smaller Δ% HMW, Δ% monomer, Δ% acidification value, and / or Δ% LMW value compared to Herceptin®.

 The formulation of the present invention comprises a combination of a stabilizer, a buffering agent, and a surfactant as described above, between trastuzumab or an antigen-binding fragment thereof by interaction with the side chain of trastuzumab or an antigen-binding fragment thereof. Storage stability is high because it blocks aggregation.

In one embodiment, the formulation may be for injection.

In one embodiment, the formulation may be for subcutaneous or intravenous injection. The formulation of the present invention contains a high concentration of trastuzumab or an antigen-binding fragment thereof and is highly concentrated, but has reduced viscosity, making it easy to manufacture, transport and store trastuzumab or an antigen-binding fragment thereof, and to reduce pain during administration. Does not cause In one embodiment, the formulation of the present invention does not contain hyaluronidase, so the possibility of side effects is low.

The formulation may further include an aqueous carrier suitable for injection. The aqueous carrier is a safe and non-toxic pharmaceutical acceptable when administered to humans, including, but not limited to, sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), sterile saline solution, Ringer's solution, dextrose Does not work.

In one embodiment, the formulations of the present invention may have an appropriate range of osmolality upon subcutaneous or intravenous injection. Osmolarity concentrations are, for example, 257 to 636, 285 to 517, 275 to 527, 255 to 547, 235 to 567, 257 to 485, 247 to 495, 227 to 515, 207 to 535, 290 to 608, 280 to 618, 260 to 638, 240 to 658, 200 to 400, 250 to 350, 270 to 330, 336 to 636, 326 to 646, 316 to 656, about 544, 534 to 554, or 524 to 564 mOsm / kg have. The osmolarity concentration can be appropriately adjusted to minimize pain that may occur during administration.

In one embodiment, the formulations of the present invention may have an appropriate range of viscosity upon subcutaneous or intravenous injection. Such viscosities can include the viscosity ranges discussed above. The viscosity range can be appropriately adjusted to minimize pain that may occur during administration.

Another aspect of the invention, one or more selected from the group consisting of at least 100 mg / mL trastuzumab or an antigen-binding fragment thereof, histidine as a buffering agent, arginine as a stabilizer, methionine and benzoate, and PS 20 as a surfactant It provides a stable pharmaceutical formulation comprising a. The content of each component in this formulation is as described above.

In one embodiment, the formulation has at least 150-300 mg / mL trastuzumab or an antigen-binding fragment thereof, 1-150 mM histidine as a buffering agent, 1-300 mM arginine as a stabilizer, 1-50 mM methionine and 10- It may be a stable pharmaceutical formulation comprising at least one selected from the group consisting of 220 mM benzoate, and 0.01 to 0.1 w / v% PS 20 as a surfactant.

In one embodiment, the formulation comprises at least 200-290 mg / mL trastuzumab or an antigen-binding fragment thereof, 1-70 mM histidine as a buffering agent, 10-220 mM arginine as a stabilizer, 5-25 mM methionine and 15- It may be a stable pharmaceutical formulation comprising at least one selected from the group consisting of 210 mM benzoate, and 0.01 to 0.07 w / v% PS 20 as a surfactant.

In one embodiment, the formulation comprises at least 220 to 270 mg / mL trastuzumab or an antigen binding fragment thereof, 10 to 50 mM histidine as a buffering agent, 30 to 200 mM arginine as a stabilizer, 10 to 20 mM methionine and 25 to It may be a stable pharmaceutical formulation comprising at least one selected from the group consisting of 200 mM benzoate, and 0.01 to 0.07 w / v% PS 20 as a surfactant.

In one embodiment, the formulation may not contain sugars, sugar alcohols and sugar acids.

The pH of the formulation is 5.0 to 7.0, 4.9 to 7.1, 4.8 to 7.2, 4.7 to 7.3, 4.5 to 7.5, 5.3 to 7.0, 5.2 to 7.1, 5.1 to 7.2, 5.0 to 7.3, 4.8 to 7.5, about 5.5, 5.4 to 5.6, 5.3 to 5.7, 5.2 to 5.8, 5.3 to 5.7, 5.2 to 5.8, 5.1 to 5.9, 5.0 to 6.0, 4.8 to 6.2, 6.0 to 7.0, 5.9 to 7.1, 5.8 to 7.2, 5.7 to 7.3, or 5.5 to 7.5 Can be

The viscosity of the formulation is 100 cP or less, 90 cP, 22 to 86 cP, 20 to 88 cP, 17 to 91 cP, 12 to 96 cP, 22 to 73 cP, 20 to 75 cP, 17 to 78 cP, 12 to 83 cP, 53-82 cP, 51-84 cP, 48-87 cP, 43-92 cP, about 30 cP, 28-32 cP, 25-35 cP, 20-40 cP, 34-73 cP, 32-75 cP , 29 to 78 cP, 24 to 83 cP, 47 to 86 cP, 45 to 88 cP, 42 to 91 cP, or 37 to 96 cP.

The osmolality of the formulations is 257 to 636, 285 to 517, 275 to 527, 255 to 547, 235 to 567, 257 to 485, 247 to 495, 227 to 515, 207 to 535, 290 to 608, 280 to 618 , 260 to 638, 240 to 658, 200 to 400, 250 to 350, 270 to 330, 336 to 636, 326 to 646, or 316 to 656 mOsm / kg.

Group 1:

Another embodiment is 150-300 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 150 mM histidine, 1 to 300 mM arginine, and 0.01 to 0.1 w / v% PS 20.

One embodiment is Trastuzumab at 190 to 300 mg / mL or an antigen binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 70 mM histidine and 50 to 250 mM arginine and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 200 to 290 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 60 mM histidine, 80 to 220 mM arginine and 0.01 to 0.1 w / v% PS 20.

One embodiment includes trastuzumab at 220-270 mg / mL or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 10 to 20 mM histidine and 100 to 200 mM arginine and 0.01 to 0.1 w / v% PS 20.

In embodiments of this group, the pH of the formulation may be between pH 5.0 and 6.0, 4.8 and 6.2, or 4.6 and 6.4. The formulation may have a viscosity of 22 to 74 cP, 20 to 76 cP, or 17 to 79 cP. The formulation may have an osmotic pressure of 285 to 517, 275 to 527, or 255 to 547 mOsm / kg.

In the above three embodiments, the liquid composition is a change amount of% HMW measured when stored at 40 ° C for 4 weeks with conventional SE-HPLC, when the antibody content is 220 to 270 mg / ml (pH 5.5). ,% HMW at Week 4-% HMW at Week 0 is about 3.2 or less, about 3.1 or less, about 3.0 or less, about 2.9 or less, about 2.8 or less, about 2.7 or less, about 2.6 or less, about 2.5 or less, about 2.4 or less , About 2.3 or less, about 2.2 or less, about 2.1 or less, about 2.0 or less, about 1.93% or less, 1.0 to 3.2%, or 1.5 to 3.1%, but is not limited thereto. The "O parking" indicates initial storage.

In the above-described three embodiments, the liquid formulation is the antibody content of 220 to 270 mg / ml (pH 5.5), the liquid composition is maintained at -70 ± 10 ℃ for 18 hours, and then left at room temperature for 1 hour After repeating the thawing process 5 times, the amount of change in% HMW measured by conventional SE-HPLC is about 0.2% or less, about 0.1% or less, about 0.08% or less, about 0.06% or less, about 0.05% or less, about 0.04 %, About 0.03% or less, about 0.02% or less, about 0.01% or less, about 0.001% or less, about 0.08% or less, 0.06 to 0.10%, 0.04 to 0.12%, or 0.02 to 0.14%.

In the above-described three embodiments, the liquid composition, when the antibody content is 220 to 270 mg / ml (pH 5.5), is stored at 40 ° C. for 4 weeks, and then the reference standard, the analysis control and the sample are 0.0488 to 100 μg / Diluted to a concentration of mL and placed in each well of a 96-well plate, the concentration of trastuzumab was measured using Nanodrop (Thermo, USA), and then trastuzumab labeled with europium (PerkinElmer, USA). After inserting the Cy5 labeled HER2 solution, the light was shielded against a 96-well plate and incubated for 60 minutes while stirring at 25 ± 1 ° C. at 500 rpm, and then excited each well at 340 nm to fluorescence emitted at 665 nm. When the energy is measured with an Envision multimode plate reader (Perkin Elmer, USA), the% relative binding activity (RBA) value may be 70% or more, 75% or more, 80% or more, 85% or more.

Group 2:

Another embodiment is 150-300 mg / mL trastuzumab or an antigen-binding fragment thereof; 1 to 150 mM histidine, 1 to 300 mM arginine, 1 to 50 mM methionine; And 0.01 to 0.1 w / v% PS 20.

In one embodiment, 210 to 300 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 70 mM histidine, 10 to 200 mM arginine, 1 to 30 mM methionine, and 0.01 to 0.1 w / v% PS 20.

One embodiment includes trastuzumab at 220-290 mg / mL or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 5 to 30 mM histidine, 100 to 200 mM arginine, 5 to 25 mM methionine, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 240 to 270 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 5 to 15 mM histidine and 100 to 150 mM arginine, 10 to 20 mM methionine, and 0.01 to 0.1 w / v% PS 20.

In embodiments of this group, the formulation may be pH 5.3 to 5.7, 5.1 to 5.9, or 4.9 to 6.1. The formulation may have a viscosity of 34 to 73, 32 to 75, or 31 to 77 cP. The formulation may have an osmotic pressure of 257 to 485, 247 to 495, or 227 to 515 mOsm / kg.

In the above-mentioned three embodiments, the liquid composition is a change amount of% HMW measured when stored at 40 ° C for 4 weeks with conventional SE-HPLC, when the antibody content is 240 to 270 mg / ml (pH 5.5). , (% HMW at Week 4 of storage)-(% HMW at Week 0) of about 2.5% or less, about 2.0% or less, about 1.65% or less, about 1.85% or less, about 2.05% or less, 1.45 to 1.85%, or It may be 1.15 to 2.15%, but is not limited thereto. The "O parking" indicates initial storage.

In the above-mentioned three embodiments, the liquid formulation, when the antibody content is 240 to 270 mg / ml (pH 5.5), the liquid composition is maintained at -70 ± 10 ° C. for 18 hours, and then left at room temperature for 1 hour. After repeating the thawing process 5 times, the amount of change in% HMW measured by conventional SE-HPLC is about 2.5% or less, 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 0.08% or less, about 0.01% or less, about 0.001% or less, about 0.06% or less, 0.04 to 0.08%, or 0.02 to 0.10%.

Group 3:

Another embodiment is 150-300 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 150 mM histidine, 1 to 300 mM arginine, 10 to 220 mM benzoate, and 0.01 to 0.1 w / v% PS 20 as surfactant.

One embodiment includes trastuzumab at 220-300 mg / mL or an antigen-binding fragment thereof; It can be a stable pharmaceutical formulation comprising 1 to 70 mM histidine and 10 to 150 mM arginine, 10 to 200 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 250 to 290 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 5 to 55 mM histidine and 20 to 110 mM arginine, 10 to 165 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 260 to 280 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 10 to 50 mM histidine, 30 to 100 mM arginine, 25 to 150 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In embodiments of this group, the formulation may be pH 5.3 to 7.0, 5.1 to 7.2, or 4.9 to 7.4. The formulation may have a viscosity of 53 to 82, 51 to 84, 48 to 87, 43 to 92, 33 to 102 cP. The formulation may have an osmotic pressure of 290 to 608, 280 to 618, 260 to 638, or 240 to 658 mOsm / kg. The formulation may not contain methionine.

In the above four embodiments, the liquid composition is a% HMW measured when stored at 40 ° C for 4 weeks with conventional SE-HPLC when the antibody content is 260 to 280 mg / mL mg / ml (pH 5.5). The amount of change, i.e., (% HMW at Week 4 of storage)-(% HMW at Week 0) is about 3.2% or less, about 3.1% or less, about 3.0% or less, about 2.9% or less, about 2.8% or less, about 2.7% Or less, about 2.6% or less, about 2.5% or less, about 2.4% or less, about 2.3% or less, and specifically, may be 2.2 to 3.2%, but is not limited thereto. The "O parking" indicates initial storage.

In the above-described four embodiments, the liquid formulation, when the antibody content is 260 to 280 mg / ml (pH 5.5), the liquid composition is maintained at -70 ± 10 ℃ for 18 hours, and then left at room temperature for 1 hour After repeating the thawing process 5 times, the amount of change in% HMW measured by conventional SE-HPLC is about 0.2% or less, about 0.1% or less, about 0.08% or less, about 0.06% or less, about 0.05% or less, about 0.04 %, About 0.03% or less, about 0.02% or less, about 0.01% or less, about 0.001% or less, about 0.08% or less, 0.06 to 0.10%, 0.04 to 0.12%, or 0.02 to 0.14%.

Group 4:

Other embodiments are 150 to 300 mg / mL trastuzumab or antigen binding fragments thereof, 1 to 150 mM histidine as a buffering agent, 10 to 220 mM benzoate as a stabilizer and 0.01 to 0.1 w / v% PS as a surfactant It can be a stable pharmaceutical formulation comprising 20.

One embodiment includes trastuzumab at 220-300 mg / mL or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 70 mM histidine, 100 to 250 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 250 to 290 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 30 to 70 mM histidine, 175 to 220 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 260 to 280 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 40 to 60 mM histidine, 185 to 215 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In embodiments of this group, the pH of the formulation may be about 5.5, 5.4 to 5.6, 5.3 to 5.7, or 5.2 to 5.8. The viscosity of the formulation may be about 30 cP, 28 to 32 cP, 25 to 35 cP, or 20 to 40 cP. The osmolarity concentration of the formulation may be about 543.5, 523 to 563, 503 to 583, or 483 to 603 mOsm / kg. The benzoate may be sodium benzoate. The concentration of PS 20 may be 0.02 to 0.06 w / v%.

Group 5:

Another embodiment is 150-300 mg / mL trastuzumab or an antigen-binding fragment thereof; 1 to 150 mM histidine, 1 to 300 mM arginine, 1 to 30 mM methionine; It may be a stable pharmaceutical formulation comprising 10 to 220 mM benzoate and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 240 to 300 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 1 to 70 mM histidine and 10 to 200 mM arginine, 1 to 50 mM methionine, 10 to 200 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 250 to 290 mg / mL trastuzumab or an antigen-binding fragment thereof; It may be a stable pharmaceutical formulation comprising 5 to 55 mM histidine and 20 to 150 mM arginine, 5 to 25 mM methionine, 50 to 165 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In one embodiment, 250 to 290 mg / mL trastuzumab or an antigen-binding fragment thereof; It can be a stable pharmaceutical formulation comprising 10 to 50 mM histidine and 30 to 100 mM arginine, 10 to 20 mM methionine, 75 to 150 mM benzoate, and 0.01 to 0.1 w / v% PS 20.

In embodiments of this group, the pH of the formulation may be 5.9 to 7.0, 5.8 to 7.1, 5.7 to 7.2, 5.6 to 7.3, or 5.5 to 7.4. The viscosity of the formulation may be 47 to 86 cP, 45 to 88 cP, 42 to 91 cP, or 37 to 96 cP. The osmolarity concentration of the formulation may be 336 to 636, 326 to 646, or 316 to 656 mOsm / kg. The benzoate may be sodium benzoate. The concentration of PS 20 may be 0.02 to 0.06 w / v%.

In the above four embodiments, the liquid composition is a% HMW measured when stored at 40 ° C for 4 weeks with conventional SE-HPLC when the antibody content is 260 to 280 mg / mL mg / ml (pH 5.5). The amount of change, i.e., (% HMW at week 4 of storage)-(% HMW at week 0) of about 3.3% or less, about 3.2% or less about 3.1% or less, about 3.0% or less, about 2.9% or less, about 2.8% or less , About 2.7% or less, about 2.6% or less, and specifically 3.2 to 2.6%, but is not limited thereto. The "O parking" indicates initial storage.

In the above-described four embodiments, the liquid formulation, when the antibody content is 260 to 280 mg / ml (pH 5.5), the liquid composition is maintained at -70 ± 10 ℃ for 18 hours, and then left at room temperature for 1 hour After repeating the thawing process 5 times, the amount of change in% HMW measured by conventional SE-HPLC is about 0.2% or less, about 0.1% or less, about 0.08% or less, about 0.06% or less, about 0.05% or less, about 0.04 %, About 0.03% or less, about 0.02% or less, about 0.01% or less, about 0.001% or less, about 0.08% or less, 0.06 to 0.10%, 0.04 to 0.12%, or 0.02 to 0.14%.

Another aspect of the present invention, a buffering agent in a solvent; And preparing a mixed solution by adding at least one selected from the group consisting of arginine, methionine and benzoate as a stabilizer; Adding trastuzumab or an antigen-binding fragment thereof to the mixed solution at least 100 mg / ml; And it provides a method for producing a stable pharmaceutical formulation comprising the step of adding a surfactant.

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

Claims (19)

1. (a) Trastuzumab at least 100 mg / ml or an antigen-binding fragment thereof;

   (b) buffering agents;

   (c) one or more selected from the group consisting of arginine, methionine and benzoate; And

   (d) A stable pharmaceutical formulation comprising a surfactant.
2. The pharmaceutical formulation of claim 1, wherein the formulation has a viscosity of 100 c or less.
3. The method according to claim 1, wherein the formulation is a pharmaceutical formulation that does not contain a sugar, sugar alcohol or sugar acid.
4. The method according to claim 1, The concentration of the trastuzumab or antigen-binding fragment thereof is a pharmaceutical formulation of 150 to 300 mg / mL.
5. The pharmaceutical formulation of claim 1, wherein the buffering agent is histidine.
6. The pharmaceutical formulation of claim 1, wherein the histidine has a concentration of 1 to 150 mM.
7. The pharmaceutical formulation of claim 1, wherein the arginine concentration is 1 to 300 mM.
8. The pharmaceutical formulation of claim 1, wherein the benzoate concentration is 10 mM or more.
9. The method according to claim 1, wherein the methionine concentration is 1 to 50 mM or more pharmaceutical formulation.
10. The pharmaceutical formulation of claim 1, wherein the surfactant is a nonionic surfactant.
11. The pharmaceutical formulation of claim 1, wherein the pH of the formulation is 4.0 to 7.5.
12. The pharmaceutical formulation of claim 1, wherein the formulation is for injection.
13. The pharmaceutical formulation of claim 1, wherein the formulation is for subcutaneous injection.
14. The method according to claim 1, wherein the formulation has a content of trastuzumab or an antigen-binding fragment thereof of 220 to 270 mg / ml (pH 5.5), and stored at 40 ° C. for 4 weeks, the reference standard, analytical control and sample are 0.0488 to Diluted to a concentration of 100 μg / mL into each well of a 96-well plate, the concentration of trastuzumab was measured using Nanodrop (Thermo, USA), and then trastuzumab labeled with europium (PerkinElmer, USA) and Cy5 labeled HER2 solution, after blocking light for 96-well plate and incubating at 25 ± 1 ° C at 500 rpm for 60 minutes, excite the sample contained in each well to 340 nm When the fluorescence energy emitted at 665 nm is measured with an Envision multimode plate reader (Perkin Elmer, USA), the% relative binding activity (RBA) value is 70% or more.
15. The method according to claim 1, wherein the formulation has a content of trastuzumab or an antigen-binding fragment thereof of 220 to 270 mg / ml (pH 5.5), and the amount of change in% HMW measured by SE-HPLC after storage at 40 ° C for 4 weeks, that is, , 4th week of storage% HMW-0th week,% HMW value is 3.2% or less Pharmaceutical preparations.
16. The method according to claim 1, wherein the liquid formulation has a content of trastuzumab or an antigen-binding fragment thereof of 220 to 270 mg / ml (pH 5.5), maintained at -70 ± 10 ° C for 18 hours, and then left for 1 hour at room temperature. After repeating the thawing process 5 times, the pharmaceutical preparation of which the amount of change in% HMW measured by SE-HPLC is 0.2% or less.
17. The method according to claim 1, wherein the formulation has a trastuzumab or antigen-binding fragment content of 220 to 270 mg / ml (pH 5.5), and stored for 4 weeks at a temperature of 40 ± 2 ° C. and a relative humidity of 75 ± 5%. A pharmaceutical formulation having a% acidification value measured after 30 or less.
18. The method according to claim 1,

    The concentration of trastuzumab or antigen-binding fragment thereof is 240 to 270 mg / mL,

    The buffering agent is 5 to 15 mM histidine,

    The stabilizer is 100 to 150 mM arginine, and 10 to 20 mM methionine,

    The surfactant is a pharmaceutical formulation of 0.01 to 0.1 w / v% PS 20.
19. Preparing a mixed solution by adding one or more of arginine, methionine or benzoate as a buffering agent and a stabilizer to the solvent;

    Adding trastuzumab or an antigen-binding fragment thereof to the mixed solution at least 100 mg / ml; And

    A method of making a pharmaceutical formulation comprising adding a surfactant.

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