Freeze-drying protective agent of antibody coupling medicine and application of freeze-drying protective agent

Abstract

The invention relates to a freeze-drying protective agent of an antibody coupling medicine and application of the freeze-drying protective agent. The freeze-drying protective agent contains a surfactant, a stabilizer, an additive and a buffer solution, the surfactant comprises poloxamer 188, the stabilizer comprises cane sugar and / or trehalose, and the additive comprises any one or a combination of at least two of arginine, methionine or EDTA (Ethylene Diamine Tetraacetic Acid). The invention provides a formula of a freeze-drying protective agent capable of stabilizing ADC molecules, the formula of the freeze-drying protective agent is simple, ADC drug molecules can be stably preserved at 40 DEG C for at least one month, and the freeze-drying protective agent has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a freeze-drying protective agent for antibody-drug conjugates and applications thereof. Background Art

[0002] Antibody drug conjugates (ADCs) are a new type of biological drug that is made by coupling a monoclonal antibody and a cytotoxic small molecule payload through a bioactive linker. This drug combines the high specificity and targeting of monoclonal antibodies with small molecules that have the ability to kill cancer cells efficiently. After entering the human body, it can concentrate the drug effect on cancer cells through the targeting ability of antibodies, effectively reducing the severe side effects of small molecule drugs when used alone, while giving full play to the ability of small molecules to eliminate cancer cells. In addition, the use of linkers with different structures can further regulate the controlled release of ADC drugs. Therefore, ADC is a new type of drug that can synergistically exert the advantages of antibody drugs and chemical drugs and has great clinical application potential.

[0003] CN111939267A discloses an antibody-drug conjugate preparation, a preparation method and an application. The solid preparation comprises an antibody-drug conjugate composed of an antibody, a linker and a drug module, a buffer, a protective agent and a surfactant. The buffer comprises one or more of a histidine buffer, a citrate buffer and a succinate buffer, the protective agent is selected from one or both of sucrose and trehalose, and the surfactant is selected from one or both of Tween 20 and Tween 80.

[0004] CN113677364A discloses a pharmaceutical composition comprising an anti-191P4D12 antibody-drug conjugate and a method of using the same, wherein the drug-antibody conjugate comprises an antibody or antigen-binding fragment thereof capable of binding to 191P4D12 coupled to one or more units of monomethyl auristatin E, and a pharmaceutically acceptable excipient comprising L-histidine, polysorbate-20, and at least one of trehalose dihydrate and sucrose.

[0005] Since the structure of antibody-drug conjugates consists of three components: antibodies, linkers, and payloads, the complexity of their surface charge and hydrophilic-hydrophobic distribution places higher demands on the selection of surfactants. The most commonly used polysorbate surfactants in ADC prescriptions can prevent the hydrophobic aggregation of ADC molecules by aggregating at the hydrophilic-hydrophobic interface, thereby effectively improving the aggregation and particle generation of ADC macromolecules under external disturbances, temperature, and freeze-thaw pressure. However, polysorbate surfactants are easily degraded and oxidized under certain conditions, which has a negative effect on the aggregation of certain ADC drugs.

[0006] In summary, in view of the problem that there are few types of surfactants in the lyoprotectants for ADCs on the market and there is a risk of degradation, how to provide a new lyoprotectant for ADCs has become one of the problems to be solved urgently in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a lyoprotectant for an antibody-drug conjugate and its application. The lyoprotectant has a simple formula and can stably preserve ADC drug molecules at 40°C for at least 1 month. The surfactant is poloxamer 188, which has a mechanism different from that of commonly used polysorbate surfactants, and provides more surfactant options for ADC lyophilized preparations.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a lyoprotectant for an antibody-drug conjugate, wherein the lyoprotectant contains a surfactant, a stabilizer, an additive and a buffer;

[0010] The surfactant includes poloxamer 188, the stabilizer includes sucrose and / or trehalose, and the additive includes any one of arginine, methionine or EDTA or a combination of at least two thereof.

[0011] Poloxamer 188 is a type of surfactant composed of a hydrophobic segment of propylene oxide in the middle and hydrophilic segments of ethylene oxide at both ends. Its antibody stabilization mechanisms mainly have the following two aspects: on the one hand, the hydrophobic end of poloxamer 188 can directly bind to the hydrophobic end of the surface of the antibody-drug conjugate macromolecule, reducing the aggregation caused by the hydrophobic area through steric hindrance; on the other hand, poloxamer 188 competitively occupies the interface, reducing the risk of exposure of the antibody-drug conjugate macromolecules at various interfaces.

[0012] The structure and mechanism of action of Poloxamer 188 are different from those of commonly used polysorbate surfactants, and it performs better than polysorbate surfactants in prescriptions and can effectively prevent the formation of aggregates, proving that it has great development potential. In addition, the degradation risk of Poloxamer 188 is lower than that of polysorbate surfactants used in ADC preparations, and its structure has good biocompatibility.

[0013] The lyoprotectant provided by the present invention can be applied to all kinds of ADC molecules, especially to ADC molecules whose toxic small molecules are Eribulin or Exatecan. ADC molecules with such small molecules have special hydrophilicity and hydrophobicity, and the commonly used polysorbate 20 and polysorbate 80 have limited effects on such ADC molecules. However, the present invention uses poloxamer 188 to increase the steric hindrance between molecules, effectively preventing the aggregation of such molecules, thereby improving the stability of ADC molecules with the small molecule structure of Eribulin or Exatecan.

[0014] The present invention adds sucrose and trehalose as stabilizers, which can synergistically improve the stability of ADC molecules and reduce the aggregation of ADC molecules. The introduction of arginine and methionine can effectively reduce the effect of protein concentration on viscosity in the preparation, reduce oxidation of ADC preparations during storage, and synergistically improve the stability of freeze-dried preparations of antibody-drug conjugates. EDTA plays an antioxidant role by chelating metal ions.

[0015] Preferably, the stabilizer also includes mannitol.

[0016] The invention improves the freeze-drying efficiency and reduces the water content in the freeze-dried preparation by adding mannitol.

[0017] Preferably, the additive comprises arginine, methionine and EDTA.

[0018] Preferably, the pH of the buffer solution is 5-6.

[0019] Preferably, the buffer comprises a histidine buffer.

[0020] Preferably, the lyophilization protectant contains 0.02-0.2% w / v surfactant, 20-60% w / v stabilizer, 100-500 mM arginine, 20-80 mM methionine, 0.005-0.02% w / v EDTA and 5-40 mM histidine buffer with a pH of 5-6.

[0021] The specific point values ​​of 0.02-0.2% mentioned above may be 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, etc.

[0022] The specific point values ​​of 20-60% mentioned above may be selected from 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60% etc.

[0023] The specific point values ​​of 100 to 500 mM mentioned above may be 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM, etc.

[0024] The specific point value of 20 to 80 mM can be selected from 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM, etc.

[0025] The specific point values ​​of 0.005% to 0.02% mentioned above may be 0.005%, 0.007%, 0.01%, 0.012%, 0.015%, 0.017% or 0.02%, etc.

[0026] The specific point values ​​of 5 to 6 mentioned above can be selected from 5, 5.2, 5.4, 5.6, 5.8 or 6, etc.

[0027] The specific point values ​​of 5 to 40 mM mentioned above can be selected from 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM or 40 mM, etc.

[0028] Preferably, the stabilizer contains trehalose and sucrose, and the mass ratio of trehalose to sucrose is 1:(1-3).

[0029] The specific point values ​​of the above (1-3) can be selected as 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8 or 3, etc.

[0030] Preferably, the stabilizer contains trehalose, sucrose and mannitol, and the mass ratio of trehalose, sucrose and mannitol is 1:(1-3):(1-3).

[0031] The specific point values ​​of the above (1-3) can be selected as 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8 or 3, etc.

[0032] In a second aspect, the present invention provides a composite solution containing an antibody-drug conjugate, wherein the composite solution contains an antibody-drug conjugate and the lyoprotectant described in the first aspect.

[0033] Preferably, the composite solution contains 5 to 30 mg / mL (eg, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL or 30 mg / mL, etc.) of antibody-drug conjugate.

[0034] Preferably, the preparation method of the composite solution comprises: mixing the antibody-drug conjugate solution with a lyophilization protectant.

[0035] Preferably, the solvent of the antibody-drug conjugate solution is a 5-40 mM histidine buffer with a pH of 5-6.

[0036] The above-mentioned 5 to 6 can be 5, 5.2, 5.4, 5.6, 5.8 or 6, etc.

[0037] The above-mentioned 5-40mM may be 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM or 40mM, etc.

[0038] Preferably, the volume ratio of the antibody-drug conjugate solution to the lyoprotectant is (3-5):1.

[0039] The above-mentioned 3 to 5 can be 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8 or 5, etc.

[0040] In a third aspect, the present invention provides a lyophilized preparation of an antibody-drug conjugate, wherein the lyophilized preparation is obtained by freeze-drying the composite solution described in the second aspect.

[0041] In a fourth aspect, the present invention provides a method for preparing the lyophilized preparation as described in the third aspect, the preparation method comprising the steps of freezing, primary drying and secondary drying.

[0042] Preferably, the freezing procedure includes: freezing at 0-10°C for 0.5-2h, freezing at -25--15°C for 1-3h, freezing at -50--40°C for 2-6h, freezing at -20-0°C for 2-6h, and freezing at -50--40°C for 2-6h.

[0043] The specific point values ​​of 0 to 10°C may be 0°C, 2°C, 4°C, 6°C, 8°C or 10°C, etc.

[0044] The specific point values ​​of 0.5 to 2h mentioned above can be selected as 0.5h, 0.7h, 0.9h, 1h, 1.1h, 1.3h, 1.5h, 1.7h, 1.9h or 2h, etc.

[0045] The specific point values ​​of -25 to -15°C mentioned above can be selected from -25°C, -23°C, -21°C, -20°C, -19°C, -17°C or -15°C, etc.

[0046] The specific point values ​​of the above 1 to 3 hours can be selected as 1h, 1.3h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.7h or 3h, etc.

[0047] The specific point values ​​of -50 to -40°C mentioned above can be selected from -50°C, -48°C, -46°C, -45°C, -44°C, -42°C or -40°C, etc.

[0048] The specific point values ​​of 2 to 6 hours mentioned above can be selected as 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h or 6h, etc.

[0049] The specific point values ​​of -20 to 0°C mentioned above can be selected from -20°C, -15°C, -12°C, -10°C, -8°C, -5°C or 0°C, etc.

[0050] The specific point values ​​of -50 to -40°C mentioned above can be selected from -50°C, -48°C, -46°C, -44°C, -42°C or -40°C, etc.

[0051] Preferably, the primary drying procedure includes: drying at -25 to -5°C and 0.1 to 0.3 mbar for 30 to 100 hours.

[0052] The specific point values ​​of -25 to -5°C mentioned above can be selected from -25°C, -20°C, -17°C, -15°C, -13°C, -10°C or -5°C, etc.

[0053] The specific point value of 0.1 to 0.3 mbar mentioned above can be selected from 0.1 mbar, 0.15 mbar, 0.2 mbar, 0.25 mbar or 0.3 mbar, etc.

[0054] The specific point value of the above 30~100h can be selected as 30h, 40h, 50h, 60h, 70h, 80h, 90h or 100h, etc.

[0055] Preferably, the secondary drying procedure includes: drying at 20-35° C. and 0.1-0.3 mbar for 10-20 hours.

[0056] The specific point values ​​of 20-35°C can be selected from 20°C, 23°C, 25°C, 27°C, 29°C, 31°C, 33°C or 35°C, etc.

[0057] The specific point value of 0.1 to 0.3 mbar mentioned above can be selected from 0.1 mbar, 0.15 mbar, 0.2 mbar, 0.25 mbar or 0.3 mbar, etc.

[0058] The specific point values ​​of the above 10 to 20 hours can be selected as 10h, 12h, 14h, 15h, 16h, 18h or 20h, etc.

[0059] In a fifth aspect, the present invention provides a reconstituted preparation of a lyophilized preparation of an antibody-drug conjugate, wherein the reconstituted preparation is obtained by mixing the lyophilized preparation described in the third aspect with a solvent.

[0060] Preferably, the solvent is water.

[0061] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] (1) The present invention provides a lyoprotectant formulation capable of stabilizing ADC molecules. The lyoprotectant formulation is simple and can stably preserve ADC drug molecules at 40°C for at least one month. The surfactant is poloxamer 188, which has a different mechanism from the commonly used surfactants of polysorbates, and provides more surfactant options for ADC lyophilized preparations.

[0064] (2) The present invention uses poloxamer 188 as a surfactant, which has a better effect of preventing the formation of aggregates, while arginine, EDTA, and methionine as additives can provide a guarantee for the stability of ADC molecules without affecting the formation of freeze-dried products. There is no precedent for freeze-dried preparations using poloxamer 188 as a surfactant in marketed ADC drugs. It is of great significance to introduce a freeze-dried protective agent using poloxamer 188 as a surfactant into the preparation system of ADC molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the starting size exclusion chromatogram of Example 1. DETAILED DESCRIPTION

[0066] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0067] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0068] The weight volume percentage (%, w / v) involved in the following content represents the number of grams (g) per 100 milliliters (100 mL), and the molar concentration (mM, mmol / L) represents the number of moles (mol) of solute contained in each liter (L) of solution.

[0069] Reagents used in the following examples:

[0070] Arginine: purchased from Merck, product number 1.01587;

[0071] Methionine: purchased from Nanning Evonik Meishi Pharmaceutical Co., Ltd.

[0072] Sucrose: purchased from Pfanstiehl, product number S-124-2-MC;

[0073] EDTA: purchased from Avantor, product number 8995;

[0074] Trehalose dihydrate: purchased from Pfanstiehl, product number T-104-4;

[0075] Mannitol: purchased from Pfanstiehl, product number M-109-7;

[0076] Poloxamer 188: purchased from Merck, product number 1.37112;

[0077] Polysorbate 20: purchased from CRODA, product number SR40606;

[0078] Polysorbate 80: purchased from CRODA, product number SR40925.

[0079] Preparation Example 1

[0080] In this preparation example, a lyophilized protective agent was prepared, which consisted of 0.1% w / v poloxamer 188, 40% w / v stabilizer, 300 mM arginine, 50 mM methionine, 0.01% w / v EDTA and 20 mM histidine buffer at pH 5.5, wherein the mass ratio of trehalose, sucrose and mannitol in the stabilizer was 1:2:2.

[0081] Preparation Example 2

[0082] In this preparation example, a lyophilized protective agent was prepared, which consisted of 0.2% w / v poloxamer 188, 20% w / v stabilizer, 500 mM arginine, 80 mM methionine, 0.005% w / v EDTA and 5 mM histidine buffer at pH 5, wherein the mass ratio of trehalose, sucrose and mannitol in the stabilizer was 1:3:1.

[0083] Preparation Example 3

[0084] In this preparation example, a lyoprotectant is prepared, which consists of 0.02% w / v poloxamer 188, 60% w / v stabilizer, 100 mM arginine, 20 mM methionine, 0.02% w / v EDTA and pH 6 40 mM histidine buffer, wherein the mass ratio of trehalose, sucrose and mannitol in the stabilizer is 1:1:3.

[0085] Preparation Example 4

[0086] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that sucrose is replaced by an equal amount of trehalose.

[0087] Preparation Example 5

[0088] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that trehalose is replaced by an equal amount of sucrose.

[0089] Preparation Example 6

[0090] This preparation example prepares a lyophilization protective agent, which differs from preparation example 1 only in that sucrose is replaced by an equal amount of sorbitol.

[0091] Preparation Example 7

[0092] This preparation example prepares a lyoprotectant, which differs from preparation example 1 only in that trehalose is replaced by an equal amount of sorbitol.

[0093] Preparation Example 8

[0094] This preparation example prepares a freeze-dried protective agent, which is different from Preparation Example 1 only in that mannitol is not added and the proportion of mannitol is distributed to other substances in proportion.

[0095] Preparation Example 9

[0096] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that arginine is replaced by an equal amount of methionine.

[0097] Preparation Example 10

[0098] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that methionine is replaced by an equal amount of arginine.

[0099] Preparation Example 11

[0100] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that methionine is replaced by an equal amount of glycine.

[0101] Preparation Example 12

[0102] This preparation example prepares a lyophilized protective agent, which differs from preparation example 1 only in that arginine is replaced by an equal amount of glycine.

[0103] Preparation Example 13

[0104] This preparation example prepares a freeze-dried protective agent, which is different from Preparation Example 1 only in that EDTA is not added and the proportion of EDTA is distributed to other substances in proportion.

[0105] Comparative Preparation Example 1

[0106] This comparative preparation example prepares a lyophilized protective agent, which differs from Preparation Example 1 only in that poloxamer 188 is not added and the proportion thereof is distributed to other components in the lyophilized protective agent in proportion.

[0107] Comparative Preparation Example 2

[0108] This comparative preparation example prepares a lyophilization protective agent, which differs from Preparation Example 1 only in that Poloxamer 188 is replaced by an equal amount of sodium chloride.

[0109] Comparative Preparation Example 3

[0110] This comparative preparation example prepares a lyoprotectant, which differs from Preparation Example 1 only in that poloxamer 188 is replaced by an equal amount of polysorbate 20.

[0111] Comparative Preparation Example 4

[0112] This comparative preparation example prepares a lyoprotectant, which differs from Preparation Example 1 only in that poloxamer 188 is replaced by an equal amount of polysorbate 80.

[0113] Comparative Preparation Example 5

[0114] This comparative preparation example prepares a lyophilized protective agent, which differs from Preparation Example 1 only in that arginine, methionine and EDTA are not added, and the proportions of the above three substances are distributed to other components in the lyophilized protective agent in proportion.

[0115] Example 1

[0116] This example prepares a lyophilized preparation of an antibody-drug conjugate, wherein the ADC molecule is mixed with the lyophilization protective agent provided in Preparation Example 1 at a volume ratio of 4:1, and the mixed solution contains 20 mg / mL ADC molecule. In a biological safety cabinet, all liquid preparations are sterile filtered through a 0.22 μm low protein adsorption filter and canned into a sterile 20 mL medium borosilicate glass anti-jump stopper injection bottle, which is half-stoppered with a chlorobutyl-coated freeze-dried rubber stopper, and the canning volume is 4 mL. Freeze-drying is performed according to the freeze-drying procedure shown in Table 1. After the freeze-dried sample is prepared, the sample is capped with an aluminum / plastic flip-open seal.

[0117] Table 1

[0118]

[0119] Example 2

[0120] This example prepares a lyophilized preparation of an antibody-drug conjugate, and the ADC molecule is mixed with the lyophilization protective agent provided in Preparation Example 2 at a volume ratio of 3:1, and the mixed solution contains 5 mg / mL ADC molecules. In a biological safety cabinet, all liquid preparations are sterile filtered through a 0.22 μm low protein adsorption filter and canned into a sterile 20 mL medium borosilicate glass anti-jump stopper injection bottle, which is half-stoppered with a chlorobutyl-coated freeze-dried rubber stopper, and the canning volume is 4 mL. Freeze drying is performed according to the freeze drying procedure shown in Table 2. After the freeze-dried sample is prepared, the sample is capped with an aluminum / plastic flip-open seal.

[0121] Table 2

[0122]

[0123] Example 3

[0124] This example prepares a lyophilized preparation of an antibody-drug conjugate, and the ADC molecule is mixed with the lyophilization protective agent provided in Preparation Example 3 at a volume ratio of 5:1, and the mixed solution contains 30 mg / mL ADC molecules. In a biological safety cabinet, all liquid preparations are sterile filtered through a 0.22 μm low protein adsorption filter and canned into a sterile 20 mL medium borosilicate glass anti-jump stopper injection bottle, which is half-stoppered with a chlorobutyl-coated freeze-dried rubber stopper, and the canning volume is 4 mL. Freeze drying is performed according to the freeze drying procedure shown in Table 3. After the freeze-dried sample is prepared, the sample is capped with an aluminum / plastic flip-open seal.

[0125] Table 3

[0126]

[0127] Embodiment 4-13

[0128] This example prepares a lyophilized preparation of an antibody-drug conjugate, which differs from Example 1 only in that the lyophilized protective agent is replaced by the lyophilized protective agent provided in Preparation Example 4-13.

[0129] Comparative Examples 1-5

[0130] This comparative example prepares a lyophilized preparation of an antibody-drug conjugate, which differs from Example 1 only in that the lyophilized protective agent is replaced by the lyophilized protective agent provided in Comparative Examples 1-5.

[0131] Test Example 1

[0132] This test example performs size exclusion chromatography analysis on the lyophilized preparation of the antibody-drug conjugate provided in Example 1-13, and the size exclusion chromatography analysis is performed again after being treated at 40°C for 4 weeks. The ADC molecule is trastuzumab conjugated to exitecan. The size exclusion chromatography analysis procedure is as follows: Detection is performed on an Agilent 1260 HPLC system equipped with a TSKgelG3000SWXL column (7.8×300mm, 5μm particle size) and a UV detector (detection wavelength: 280nm). The sampler temperature is set to 5±3°C, and the column oven temperature is set to 25±3°C. The mobile phase is 50mM phosphate buffer and 300mM sodium chloride, the pH value is 6.8±0.1, and the flow rate is set to 1.0mL / min. 100μg of each sample is injected into an HPLC vial. The detection wavelength is set to 280nm and the running time is 20 minutes. Finally, all raw data are processed using chromatographic data analysis software (Empower 3, Waters Corporation). The results are shown in Table 4. The initial size exclusion chromatogram of Example 1 is shown in Table 4. Figure 1 shown.

[0133] Table 4

[0134]

[0135] From Table 4 we can get:

[0136] (1) Comparing Example 1 with Examples 4-7, it can be seen that sucrose and trehalose have the ability to synergistically protect ADC molecules. When the two components are added at the same time, the drug in Example 1 still has good stability after being stored at 40° C. for 4 weeks. Removing or replacing any one of the components will reduce the protective effect of the lyoprotectant;

[0137] (2) Comparing Example 1 with Examples 9-12, it can be seen that arginine and methionine can synergistically improve the stability of the ADC lyophilized preparation. When both components are added at the same time, the drug in Example 1 still has good stability after being stored at 40° C. for 4 weeks. Removing or replacing any one component will reduce the protective effect of the lyoprotectant.

[0138] (3) Comparing Example 1 with Example 13, it can be seen that the addition of EDTA can improve the stability of the ADC lyophilized preparation.

[0139] Test Example 2

[0140] In this test example, the lyophilized preparations of antibody-drug conjugates provided in Example 1 and Comparative Examples 1-5 were subjected to initial size exclusion chromatography analysis with reference to Test Example 1. The ADC molecule was trastuzumab conjugated to eribulin. The results are shown in Table 5.

[0141] Table 5

[0142] Group Main peak% Polymer% Oligomer% Example 1 99.4 0.6 ND Comparative Example 1 96.2 3.8 ND Comparative Example 2 96.0 4.0 ND Comparative Example 3 98.5 1.5 ND Comparative Example 4 97.4 2.5 0.1 Comparative Example 5 99.2 0.8 ND

[0143] From Table 5 we can get:

[0144] (1) Comparison of Example 1 with Comparative Examples 1-2 shows that poloxamer 188 is an important component in ADC lyoprotectants, and the addition of poloxamer 188 can significantly reduce the aggregation of ADC molecules;

[0145] (2) Comparison of Example 1 with Comparative Examples 3-4 shows that the use of Poloxamer 188 as a surfactant has a better technical effect than polysorbates and is more conducive to reducing the aggregation of ADC molecules;

[0146] (3) Comparing Example 1 with Example 5, it can be seen that arginine, methionine and EDTA help to reduce the aggregation of ADC molecules.

[0147] Test Example 3

[0148] This test example analyzes the moisture content of the freeze-dried preparations of antibody-drug conjugates provided in Examples 1 and 8, and analyzes the moisture content of the freeze-dried preparations again after treatment at 40°C for 4 weeks. The test method is to use a MettlerToledo C30S Karl Fischer titrator to test the moisture content of the freeze-dried samples. Before testing the sample, the reliability of the test is confirmed by testing the water standard sample and the corresponding volume of the empty bottle. Subsequently, a certain volume of anolyte is extracted from the electrolytic cell and added to the freeze-dried sample. After sufficient mixing, part of the anolyte is extracted to measure its moisture content. After the measurement, the empty bottle and the rubber stopper are cleaned and dried. During the process, the empty bottle, the empty bottle containing the anolyte, the sample bottle containing the powder cake, the sample bottle containing the powder cake and the anolyte, and the cleaned sample bottle are weighed. The moisture content of the powder cake is calculated according to the total weight of the powder cake and the anolyte, the net weight of the powder cake and the average weight of the anolyte. The ADC molecule is trastuzumab conjugated to ixetine. The results are shown in Table 6. Mannitol is not added in Example 8, and the moisture content in the freeze-dried preparation is high.

[0149] Table 6

[0150]

[0151]

[0152] In summary, the present invention provides a lyoprotectant for an antibody-drug conjugate using poloxamer 188 as a surfactant. The lyoprotectant has a simple formula, can stably preserve ADC drug molecules at 40°C for at least 1 month, and has a better effect of preventing the formation of aggregates. Arginine, methionine and EDTA as additives can provide a guarantee for the stability of ADC molecules without affecting the formation of freeze-dried products.

[0153] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A lyoprotectant for an antibody-drug conjugate, characterized in that: The lyophilization protective agent contains a surfactant, a stabilizer, an additive and a buffer; The surfactant includes poloxamer 188, the stabilizer includes sucrose and / or trehalose, and the additive includes any one of arginine, methionine or EDTA or a combination of at least two thereof.

2. The lyoprotectant according to claim 1, characterized in that The stabilizer also includes mannitol; Preferably, the pH of the buffer is 5 to 6; Preferably, the buffer comprises a histidine buffer.

3. The lyoprotectant according to claim 1 or 2, characterized in that The lyophilization protective agent contains 0.02-0.2% w / v surfactant, 20-60% w / v stabilizer, 100-500 mM arginine, 20-80 mM methionine, 0.005-0.02% w / v EDTA and 5-40 mM histidine buffer with a pH of 5-6; Preferably, the stabilizer contains trehalose and sucrose, and the mass ratio of trehalose to sucrose is 1:(1-3); Preferably, the stabilizer contains trehalose, sucrose and mannitol, and the mass ratio of trehalose, sucrose and mannitol is 1:(1-3):(1-3).

4. A composite solution containing an antibody-drug conjugate, characterized in that: The composite solution contains an antibody-drug conjugate and the lyophilization protectant according to any one of claims 1 to 3.

5. The composite solution according to claim 4, characterized in that: The composite solution contains 5 to 30 mg / mL of antibody-drug conjugate; Preferably, the preparation method of the composite solution comprises: mixing the antibody-drug conjugate solution with a lyophilization protectant; Preferably, the solvent of the antibody-drug conjugate solution is a 5-40 mM histidine buffer with a pH of 5-6; Preferably, the volume ratio of the antibody-drug conjugate solution to the lyoprotectant is (3-5):

1.

6. A lyophilized preparation of an antibody-drug conjugate, characterized in that: The freeze-dried preparation is obtained by freeze-drying the composite solution according to claim 4 or 5.

7. A method for preparing the lyophilized preparation according to claim 6, characterized in that: The preparation method comprises the steps of freezing, primary drying and secondary drying.

8. The preparation method according to claim 7, characterized in that: The freezing procedure includes: freezing at 0-10°C for 0.5-2h, freezing at -25--15°C for 1-3h, freezing at -50--40°C for 2-6h, freezing at -20-0°C for 2-6h, and freezing at -50--40°C for 2-6h.

9. The preparation method according to claim 7 or 8, characterized in that: The primary drying procedure includes: drying at -25 to -5°C and 0.1 to 0.3 mbar for 30 to 100 hours; Preferably, the secondary drying procedure includes: drying at 20-35° C. and 0.1-0.3 mbar for 10-20 hours.

10. A reconstituted preparation of a lyophilized antibody-drug conjugate preparation, characterized in that: The reconstituted preparation is obtained by mixing the lyophilized preparation according to claim 6 with a solvent; Preferably, the solvent is water.

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