Anti-Her2 antibody drug conjugate pharmaceutical preparation

Abstract

The present invention relates to a pharmaceutical formulation comprising a mixture of a non-reducing sugar, a recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate, and a surfactant. The pharmaceutical preparation has the characteristics of reducing particulate matters, reducing the formation of aggregates, improving the stability of a conjugate, improving the appearance of freeze-dried powder and the like.

Description

This invention application is a divisional application based on an invention patent application with an application date of March 25, 2020, application number 202080009504.3 (international application number PCT / CN2020 / 081138) and name “Anti-Her2 Antibody Drug Conjugate Pharmaceutical Preparation”. Technical Field The invention relates to an anti-Her2 antibody-drug conjugate pharmaceutical preparation, belonging to the field of anti-tumor drugs. Background Art

[0001] Tumor-targeted biological therapy has received increasing attention in cancer treatment research. Among them, monoclonal antibodies have high target specificity and low side effects, but their efficacy is limited when used alone. Currently, the most successful anti-tumor monoclonal antibody drugs are targeting lymphocyte tumors, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic neoplasm (CLL), etc.

[0002] ErbB2, also known as Her2 / neu, is the second member of the EGFR family. ErbB2 exerts its biological effects by forming heterodimers with the other three members of the EGFR family. The neu gene encoding ErbB2 was first isolated from rat neuroblastoma. The homologous gene of the neu gene in human somatic cells, called Her2, is located on the long arm of chromosome 17 (17q21.1). The encoded product ErbB2 consists of 1255 amino acids with a molecular weight of approximately 185kDa, of which positions 720-987 belong to the tyrosine kinase activity domain. In addition to exerting its effects through the PI3K and MAPK signaling pathways, ErbB2 can also reduce the expression of cyclin D and c-myc, thereby reducing the expression of the cyclin-dependent kinase (cdk) inhibitor p27kipl. The activity of cdk2 is inhibited, leading to cell proliferation. As research continues to expand and deepen, it has been found that HER2 is expressed and overexpressed in a variety of tumors. Therefore, effective drugs targeting HER2 are urgently needed in the clinic to treat malignant tumors. Currently, there are three monoclonal antibodies targeting Her2 on the market (see Table 1).

[0003] Table 1 FDA-approved Her2-targeting monoclonal antibody drugs

[0004] Based on the targeting characteristics of antibodies, a new generation of biological targeted drugs, antibody drug conjugates (ADC), have emerged. ADC consists of three parts: antibodies, cytotoxins, and connectors that connect the two. After the monoclonal antibody is coupled to the cytotoxin, the antibody drug conjugate uses the targeting of the monoclonal antibody to specifically identify the receptors on the surface of cancer cells, bind to the receptors, and then enter the cell, using the protease in the cell to release cytotoxins to prevent cancer cell proliferation and kill cancer cells. The existing technology generally uses mammalian cell culture to produce and express antibodies, and the highly purified antibodies are coupled with the cytotoxin MMAE through a connector to obtain an antibody drug conjugate (ADC). Antibody drug conjugation technology integrates small molecule toxin drugs with biological proteins, combining the advantages of both, becoming a new generation of therapeutic products, greatly enhancing the efficacy while reducing toxic side effects. Currently, the US FDA has approved four ADC drugs for marketing (see Table 2).

[0005] Table 2 Four ADC marketed drugs

[0006] Like other biomacromolecule drugs, ADC drugs are prone to degradation such as oxidation, deamidation and fragmentation, as well as the formation of particles and aggregates. In addition, drug conjugation itself can reduce stability and change the physicochemical properties of antibodies. For example, the conjugation of DM1 to the anti-HER2 antibody trastuzumab causes the CH2 domain of the antibody to lose stability (Document 1: Physicochemical Stability of the Antibody-Drug Conjugate Trastuzumab-DM1: Changes due to Modification and Conjugation Processes. Aditya A. Wakankar et al., Bioconjugate Chemistry 2010: 21 (9), 1588-1595). ADC drugs are usually hydrophobic, and ADC as a whole may be more insoluble than unconjugated antibodies, so it becomes easier to aggregate, form particles and surface adsorption. In order to provide ADC drugs that are stable during transportation and storage, it is necessary to carefully select the various excipient components in the drug preparation. The excipient components of currently marketed ADC drugs are shown in Table 3. Since the types of excipient ingredients used in currently marketed ADC preparations are still very limited, while there are many options for related preparation ingredients, developing an ADC excipient preparation combination that is stable during transportation and storage involves screening a large number of excipient preparation ingredients and determining appropriate concentrations, which requires a lot of effort and a long time.

[0007] Table 3 Excipients of listed ADC drugs

[0008] It can also be seen from the excipient components of the above four ADC preparations that each preparation has its own uniqueness. Due to the fragile stability and structural complexity of monoclonal antibody drugs, the manufacture and storage of such drugs are extremely challenging. Due to the heterogeneous structure of antibodies, especially the complementary determining region (CDR) and Fc glycosylation, the formulation development of different monoclonal antibodies needs to be carried out on a case-by-case basis (Reference 2: Monoclonal antibodies: formulations of marketed products and recent advances in novel delivery system, Yanan Cui et al., Drug Development and Industrial Pharmacy, Vol. 43, No. 4, pp. 519-530, 2017). ADC drugs are further involved in coupling and toxin molecules, so their formulation development is more unique.

[0009] The patent application (CN105008398A or WO2015074528A1) discloses a humanized RC48 antibody-drug conjugate, wherein the humanized RC48 antibody is a monoclonal antibody targeting Her2 (antibody secreted by or derived from hamster ovary cells (CHO cells) deposited in the China Center for Type Culture Collection with the deposit number C2013170), and the poison is selected from Monomethyl Auristatin E (MMAE), which is a derivative of sea dolitoxin peptide. The linker is Maleimido-Caproyl-Valine-Citrulline-p-AminoBenzyloxy (mc-VC-PAB), and the monoclonal antibody and MMAE in the recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate are connected to the linker via cysteine. The drug has achieved good therapeutic effects on Her2-positive tumors.

[0010] Preliminary tests have shown that the recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate obtained by coupling has poor solubility in conventional buffers after purification, and insoluble particles visible to the naked eye will appear, which does not meet the standards of injection solutions. However, if the clinically effective therapeutic dose is to be achieved, the protein concentration needs to reach 5 mg / ml or more. Therefore, how to solve the problem of insoluble particles while ensuring a certain guaranteed concentration requires further research. In addition, when the protein solution is vacuum freeze-dried, a lyoprotectant needs to be added to protect the protein from being destroyed during the freeze-drying process, while ensuring that the freeze-dried powder has a good appearance. In order to avoid the slow degradation of protein freeze-dried powder during long-term storage, an appropriate protein stabilizer needs to be added. How to determine the appropriate protein stabilizer also requires development and determination through a large number of experiments.

[0011] Therefore, the purpose of the present invention is to obtain an excipient combination of anti-Her2 monoclonal antibody-MMAE conjugate by conducting a large-scale screening and concentration range study of excipients available for biological preparations, and to achieve the following technical effects: the anti-Her2 monoclonal antibody-MMAE conjugate can be well dissolved before and after lyophilization, and the insoluble particles and visible foreign matter meet the standards of human injection solution, and at the same time, the conjugate can remain stable for a long time during the lyophilization and storage process, and is not prone to aggregation or degradation after reconstruction, so as to maintain good biological activity. Summary of the invention

[0012] The present invention provides an aqueous liquid pharmaceutical preparation of an antibody-drug conjugate, the preparation comprising an antibody-drug conjugate, a non-reducing sugar, an amino acid, and a solubilizing agent; the non-reducing sugar is selected from mannitol, sucrose, trehalose or a combination thereof; the amino acid is selected from histidine, alanine, arginine, glycine, glutamic acid or a combination thereof; the solubilizing agent is selected from glycerol, Tween80 or a combination thereof.

[0013] In some embodiments, the mannitol concentration is: 100-300mmol / L, preferably 190-300mmol / L, more preferably 200-260mmol / L, most preferably 240-260mmol / L; the sucrose concentration is: 0-100mmol / L, preferably 40-100mmol / L, more preferably 60-100mmol / L, most preferably 40-60mmol / L.

[0014] In some embodiments, the histidine is histidine hydrochloride, and its concentration is: 0-100mmol / L, preferably 5-50mmol / L, more preferably 5-20mmol / L, and most preferably 10mmol / L; the arginine is arginine hydrochloride, and its concentration is: 0-160mmol / L, preferably 20-100mmol / L, more preferably 30-90mmol / L, and most preferably about 35mmol / L.

[0015] In some embodiments, the glycerol content is: 0-1%, preferably 0.2-0.5% (w / v); the mass percentage of Tween 80 is: 0-0.02% (w / v).

[0016] Furthermore, the antibody in the antibody-drug conjugate is an anti-HER2 monoclonal antibody; the drug conjugated to the antibody is MMAE or MMAF and its derivatives or DM1, DM4 and their derivatives.

[0017] Further, the anti-HER2 antibody drug conjugate is anti-HER2 monoclonal antibody-vc-MMAE, wherein the anti-HER2 monoclonal antibody and MMAE are connected via a linker vc, wherein the linker is combined with the free thiol groups exposed after reduction of the antibody via its thiol groups, wherein the structure after the linker and MMAE are connected is:

[0018]

[0019] Furthermore, the anti-HER2 monoclonal antibody comprises a heavy chain and a light chain. (i) the heavy chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively; and / or (ii) the light chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively;

[0020] Furthermore, the monoclonal antibody is preferably a chimeric antibody or a humanized antibody.

[0021] In some embodiments, the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5-30 mg / ml.

[0022] In some embodiments, the non-reducing sugar is 240-260 mmol / L mannitol and / or 40 mmol / L-60 mmol / L sucrose, the amino acid is 8-12 mmol / L histidine hydrochloride, and the dissolution agent is 0-0.02% (w / v) Tween80.

[0023] In some embodiments, the non-reducing sugar is about 260 mmol / L mannitol and about 40 mmol / L sucrose, the amino acid is about 10 mmol / L histidine hydrochloride, the concentration of the antibody drug conjugate is about 10 mg / ml, and the dissolution agent is about 0.02% (w / v) Tween 80.

[0024] In some embodiments, the non-reducing sugar is about 240 mmol / L mannitol and about 60 mmol / L sucrose, the amino acid is about 10 mmol / L histidine hydrochloride, the concentration of the antibody drug conjugate is about 10 mg / ml, and the dissolution agent is about 0.02% (w / v) Tween 80.

[0025] Furthermore, the pH of the preparation is 4.5-7, preferably 5.6-6.8, more preferably 5.6-6.5, 5.6-6.4, 5.6-6.3, 6.1-6.4, 6.1-6.3.

[0026] The pH of the formulation was adjusted using NaOH or hydrochloric acid.

[0027] In some embodiments, the lyophilized pharmaceutical preparation is obtained by freeze-drying the above-mentioned aqueous liquid pharmaceutical preparation.

[0028] Furthermore, the aqueous liquid drug preparation before drug lyophilization contains about 260mmol / L mannitol, about 40mmol / L sucrose, about 10mmol / L histidine hydrochloride, about 0.02% (w / v) Tween 80, about 10mg / ml anti-HER2 monoclonal antibody-vc-MMAE, and the pH is 5.6-6.8.

[0029] Furthermore, the aqueous liquid drug preparation before drug lyophilization contains about 240mmol / L mannitol, about 60mmol / L sucrose, about 10mmol / L histidine hydrochloride, 0.02% Tween 80, about 10mg / ml anti-HER2 monoclonal antibody-vc-MMAE, and pH is 5.6-6.8.

[0030] Furthermore, the non-reducing sugars contained in the aqueous liquid drug preparation before drug lyophilization are mannitol and sucrose, and their concentrations are about 47.36 mg / ml and about 13.69 mg / ml, respectively; the amino acid is histidine hydrochloride, and its concentration is about 2.10 mg / ml; the solubilizing agent is Tween 80, and its content is about 0.02% (w / v).

[0031] Furthermore, the non-reducing sugars contained in the aqueous liquid drug preparation before drug lyophilization are mannitol and sucrose, and their concentrations are about 43.72 mg / ml and about 20.54 mg / ml respectively, the amino acid is histidine hydrochloride, and its concentration is about 2.10 mg / ml, and the solubilizing agent is Tween 80, and its content is about 0.02% (w / v).

[0032] The present invention further provides the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for treating a disease caused by abnormal expression of Her2, wherein the disease caused by abnormal expression is further preferably cancer; the cancer is further preferably Her2-positive cancer; the Her2-positive cancer is further preferably breast cancer, ovarian cancer, gastric cancer, urothelial carcinoma, gastroesophageal cancer, esophageal cancer, endometrial cancer, lung cancer or bladder cancer (see document 3 Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications, Nida Iqbal and Naveed Iqbal, Molecular Biology International, Volume 2014, Article ID 852748; document 4: CN201810998055.4).

[0033] The present invention further provides a method for preparing an antibody-drug conjugate pharmaceutical preparation, comprising: (1) preparing any of the above preparations; (2) Evaluate the stability of the antibody drug conjugate in the formulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : After purification, naked antibody SDS-PAGE electrophoresis, wherein, 1-2: antibody non-reducing electrophoresis 3-4: Antibody reduction electrophoresis, Figure 1 The results showed that the purity of the monoclonal antibody obtained in the experiment met the requirements for further experiments. Require;

[0035] Figure 2 : 10% SDS-PAGE electrophoresis of ADC after coupling, wherein Na: non-reducing electrophoresis; Nb: non-reducing electrophoresis after antibody is reduced by TCEP; Ra: RC48-vc-MMAE reduction electrophoresis; Rb: reduction electrophoresis after TCEP antibody reduction; Figure 2 The results showed that the purity of the ADC obtained after coupling met the requirements of further experiments; DETAILED DESCRIPTION

[0037] Example 1: Antibody Purification

[0038] Based on the related methods disclosed in the examples of the patent application (CN105008398A or WO2015074528A1), the mouse monoclonal antibody mRC48 and the related humanized antibody RC48 were obtained, which contained the human IgG1 κ heavy chain constant region and heavy chain variable region RC48-VH, and the human IgG1 κ light chain constant region and light chain variable region RC48-VL, and the above-amplified fragments were subcloned into the expression vector pcDNA3.0. The constructed different plasmids were transfected into suspension CHO cells (Invitrogen). Cultivate under standard conditions, and when the nutrients in the culture medium are exhausted and the cells no longer grow, place the tank, separate the cells by centrifugation or filtration, and take the supernatant. The antibody protein is present in the supernatant, and the first step of purification is carried out on a Protein A affinity chromatography column. The eluted target protein is purified by a cationic filler chromatography column in the second step, and the target protein peak is collected. Then, the third step of purification is carried out on the third column in the target protein penetration mode. After the purified protein passes the various index tests and is qualified, it is then ultrafiltered and concentrated to a protein concentration of about 20-30 mg / ml. This is the antibody protein stock solution, which can be stored at -80°C for a long time. The CDR sequence of the RC48 antibody is as follows: Table 4 CDR sequences of RC48 antibody Heavy chain (VH) Light chain (VL) CDR1 DYYIH (SEQ ID NO. 1) KASQDVGTAVA (SEQ ID NO. 4) CDR2 RVNPDHGDSYYNQKFKD(SEQ ID NO.2) WASIRHT (SEQ ID NO.5) CDR3 ARNYLFDHW (SEQ ID NO. 3) HQFATYT (SEQ ID NO.6) Example 2: Conjugation of Antibodies and MMAE

[0039] Conjugation of humanized antibody RC48 with drug molecules

[0040] Dissolve the mother solutions of TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (Diethylene triaminepentacetate acid) in the coupling buffer, dilute with the coupling buffer, and mix with the monoclonal antibody at a volume ratio of 1:1 (v:v=1:1). The final molar ratio of TCEP to antibody is 1.9:1, and the final concentration of DTPA is 1mmol / L. Stir and react at 25℃ for 2.5h. TCEP reduction has good reproducibility, and the number of free thiol groups after reduction can reach 3.5-4.5.

[0041] After TCEP reduction, the antibody can be directly coupled. Prepare 10mmol / L drug (vc-MMAE, vc-MMAF, mc-MMAF) dissolved in DMSO (dimethyl sulfoxide), slowly add the drug according to the molar ratio of drug to thiol of 1.1:1, and stir at 25℃ for 2h. Use DTNB method to detect the free thiol concentration (close to 0) at 412nm, purify to remove residual unreacted drugs and free small molecules such as DMSO, and detect the coupling by SDS-PAGE electrophoresis, SEC, and HPLC. The coupling reaction has good repeatability, and the opened free thiol can be coupled completely, with a coupling degree of 3.5-4.5. Example 3: Effect of pH on the recombinant humanized anti-Her-2 monoclonal antibody-MMAE conjugate (RC48-vc -MMAE) dissolution and purity

[0042] Experimental conditions: 2g of recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate protein for injection Ultrafiltration and dialysis into 0.1mol / L citric acid, 0.02mol / L tris(hydroxymethyl)aminomethane, 0.02mol / L sodium dihydrogen phosphate, 0.15mol / L sodium chloride buffer, the dialysis multiple is not less than 10 4 times, and divided into 13 portions on average. Each portion was concentrated to a protein concentration of 10 mg / ml using a centrifugal ultrafiltration tube with a pore size of 30 kD. The pH value was adjusted to 4.2, 4.6, 5.0, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, and 7.4 using hydrochloric acid or sodium hydroxide solution, and the clarity was observed and the concentration change was detected (the results are shown in Table 1). The samples were filtered into sterile 20 ml vials in a clean bench using a sterile syringe filter with a pore size of 0.22 microns, 3 vials for each pH value, sealed with sterile rubber stoppers, and covered with aluminum caps. Stored at 25°C, samples were taken with a sterile syringe at 0 hours, 1 day, 3 days, and 7 days, and SEC-HPLC purity tests were performed (the results are shown in Table 6).

[0043] The study found that changing the pH of the protein under experimental conditions has a great influence on the solubility of the protein. The protein precipitates below pH 5.4, and has good solubility above pH 5.6. The protein aggregates do not increase after 3 days of storage below pH 6.8, but increase slightly after 7 days of storage. Above pH 7.0, the aggregates increase with time, and the higher the pH, the more the aggregates increase (see Table 5 and Table 6 for specific results).

[0044] Table 5 Clarity and concentration results

[0045] Table 6 Purity results Example 4: Screening of excipients

[0046] Through a large amount of information analysis and experimental screening in the early stage, excipients such as sucrose, mannitol, glycerol, histidine, arginine, polysorbate 80 (i.e. Tween 80) were preliminarily determined as further screening candidate excipients, and the following tests were further carried out.

[0047] Table 7 Screening of RC48-vc-MMAE excipients for injection

[0048] Table 8 Protein clarity of RC48-vc-MMAE conjugate for injection

[0049] Note: “-” means the number of white dots is less than 3, “+” means the number of white dots is between 3 and 5, and “++” means the number of white dots is greater than 5.

[0050] Table 9 Purity of RC48-vc-MMAE protein for injection Recipe Number 0 hour (%) 1 day (%) 3 days (%) 7 days (%) A1 99.5 99.2 99.0 98.2 A 2 99.6 99.1 98.8 98.3 A 3 99.5 99.6 99.5 99.5 A 4 99.7 99.6 99.6 99.6 A 5 99.4 99.1 98.7 98.2 A 6 99.5 98.9 98.6 98.3 A 7 99.6 99.0 98.6 98.0 A 8 99.5 98.9 98.3 98.2

[0051] From the above results, it can be seen that adding arginine has no obvious effect on improving the visible foreign matter of protein. Adding sucrose, glycerol and polysorbate 80 can improve the situation of protein. Therefore, it is chosen to add an appropriate amount of sucrose to the formulation to protect the protein, and to add glycerol and polysorbate 80 to promote the dissolution of the protein. Example 5: Vacuum freeze drying method

[0052] The protein stock solution was taken out from the -80℃ refrigerator and melted, and accurately diluted with "1× preparation buffer" to a protein concentration of 10 mg / ml. The solution was divided into sterile, pyrogen-free standard 20ml lyophilization vials, 6ml each, and vacuum freeze-dried.

[0053] Freeze-drying conditions:

[0054] Pre-freezing: temperature -45℃, time 5 hours;

[0055] Primary drying: temperature -26°C, time 40 hours, vacuum degree 10-15Pa;

[0056] Secondary drying: temperature 25°C, time 10 hours, vacuum degree 10-15Pa.

[0057] After freeze-drying, the rubber stopper is pressed under vacuum and the aluminum cap is fastened after the sample is taken out of the freeze dryer. Example 6: Formulation Screening

[0058] The appearance and molding stability of the freeze-dried powder of the formulation preparation were examined according to the formulation designed in Table 10. The freeze-dried samples were stored at 4°C and 37°C, and the appearance was observed at 0, 1, 3, and 7 days, respectively, to screen out the formulation with better appearance and molding. The results are shown in Table 11.

[0059] Table 10 RC48-vc-MMAE preparation buffer solution screening table for injection

[0060] Table 11 Freeze-dried powder appearance inspection results

[0061] Note: 1) "--" means that the mass has shrunk to less than half of its volume before freeze-drying; "-" means that there is shrinkage, but the volume of the mass is greater than half of the volume before freeze-drying; "+" means that only the edge part has slight shrinkage, and the volume of the mass is basically the same as before freeze-drying; "++" means that there is no shrinkage at all, and the volume of the mass is the same as before freeze-drying. 2) Appearance qualification standard of freeze-dried powder: uniform color, dense pores, and the volume and shape before freeze-drying remain basically unchanged, with a block or sponge-like mass structure. It can be seen from Table 11 that the formulation containing glycerol shows slight shrinkage after being placed at 37°C for one day, and the appearance shrinks and collapses significantly after being placed for seven days, so glycerol is excluded as an excipient type for ADC preparations. Example 7. Determination of freeze-dried powder formulation and stability study

[0062] The appearance inspection of freeze-dried powder eliminated formulations B7-B12, and further excipient screening (formulas B1-B6) was carried out through moisture content inspection, visible foreign matter and insoluble particles inspection and sample stability inspection.

[0063] The lyophilized powder of each formula was stored at 37, 25 and 4°C, and samples were taken at different times for inspection of SDS-PAGE, reversed-phase HPLC, ligand binding method, biological activity, water content, appearance, pH value, visible foreign matter and insoluble particles.

[0064] Table 12 Freeze-dried powder appearance inspection results Recipe Number B1 B2 B3 B4 B5 B6 Appearance ++ ++ ++ ++ ++ ++

[0065] Note: "--" means that the mass has shrunk to less than half of its volume before freeze-drying; "-" means that there is shrinkage, but the volume of the mass is more than half of that before freeze-drying; "+" means that only the edge has slight shrinkage, and the volume of the mass is basically the same as before freeze-drying; "++" means that there is no shrinkage at all, and the volume of the mass is the same as before freeze-drying.

[0066] As can be seen from Table 12, the appearance of the freeze-dried powders of formulations B1-B6 all meet the requirements.

[0067] Three samples were taken for each formula, and the moisture content was determined according to the prescribed method. The average value was taken and the results are shown in Table 12.

[0068] Table 12 Freeze-dried powder moisture content determination results Recipe Number B1 B2 B3 B4 B5 B6 Water content (%) 0.658 0.586 0.557 0.606 0.571 0.560

[0069] The moisture contents of formulas B1, B2, B3, B4, B5, and B6 are all less than 3%, which are qualified.

[0070] Five tubes were randomly sampled for each formula and reconstituted according to the prescribed method to check for visible foreign matter. Three of the tubes were selected and the insoluble particles were determined according to the prescribed method. The results are shown in Table 13.

[0071] Table 13 Inspection results of visible foreign matter and insoluble particles after reconstitution of lyophilized powder

[0072] From the above results, it can be seen that the visible foreign matter and insoluble particles inspection showed that formulas B1, B3, and B5 did not meet the requirements and were eliminated.

[0073] Table 14 Stability test results after storage at 37℃ for 1 month

[0074] Table 15 Stability test results after 3 months storage at 25℃

[0075] Table 16 Stability test results after 6 months storage at 4℃ Description: Appearance standard: "--" indicates that the mass has shrunk to less than half of its volume before freeze-drying; "-" indicates that there is shrinkage, but the volume of the mass is greater than half of that before freeze-drying; "+" indicates that only the edge has slightly shrunk, and the volume of the mass is basically the same as before freeze-drying; "++" indicates that there is no shrinkage at all, and the volume of the mass is the same as before freeze-drying.

[0076] According to the above results, it can be seen that the stability of formula B2 and formula B4 at different temperatures (4°C, 25°C, 37°C) is good, and the insoluble particles of formula B6 are too much and eliminated. Through the above tests, formula B2 and formula B4 have reached excellent levels in the appearance inspection of lyophilized powder, the determination of the moisture content of lyophilized powder, the inspection of visible foreign matter and insoluble particles after the lyophilized powder is reconstituted, and the stability.

[0077] Based on the above experimental evidence, it can be seen that the selection of specific excipients in the major categories of non-reducing sugars, amino acids, and solubilizers has an unpredictable effect on the final preparation. For the Her2 monoclonal antibody drug conjugate of the present invention, a large number of tests are required to detect various performance aspects in order to finally obtain a good preparation combination scheme. For example, the addition of arginine has no obvious effect on improving the visible foreign matter of the protein. The addition of sucrose, glycerol, and polysorbate 80 can all improve the aggregation of proteins; however, the addition of glycerol will make the reconstituted aqueous solution of the freeze-dried product prone to shrinkage and obvious collapse after reconstitution. In addition, too high a sucrose concentration or too low a mannose concentration will lead to an increase in insoluble matter after reconstitution. These results are difficult for those skilled in the art to predict before conducting relevant experiments, and because ADC preparations involve the combined use of multiple excipients and also involve factors such as long-term stability testing, it is quite difficult to develop an ADC preparation. Through a large number of experiments, we have determined a combination of Her2 ADC preparations with excellent performance in all aspects. The anti-Her2 monoclonal antibody-MMAE conjugate can dissolve well before and after lyophilization, and the insoluble particles and visible foreign matter meet the standards for human injection. At the same time, the conjugate can remain stable for a long time during the lyophilization and storage process, especially when placed for a long time under high temperature conditions of 25°C or 37°C, it still maintains good stability. The ADC preparation is not prone to aggregation or degradation after reconstruction, and maintains good biological activity.

[0078] The present disclosure relates to the following embodiments: 1. An aqueous liquid pharmaceutical preparation of an antibody drug conjugate, the preparation comprising an antibody drug conjugate, a non-reducing sugar, an amino acid, and a solubilizing agent; the non-reducing sugar is selected from mannitol, sucrose, trehalose or a combination thereof; the amino acid is selected from histidine, alanine, arginine, glycine, glutamic acid and the hydrochlorides of the above amino acids or a combination thereof; the solubilizing agent is selected from glycerol, Tween80 or a combination thereof. 2. The aqueous liquid pharmaceutical preparation of item 1, wherein the mannitol concentration is: 100-300mmol / L, preferably 190-300mmol / L, more preferably 200-260mmol / L, most preferably 240-260mmol / L; the sucrose concentration is: 0-100mmol / L, preferably 40-100mmol / L, more preferably 60-100mmol / L, most preferably 40-60mmol / L. 3. The aqueous liquid pharmaceutical preparation described in item 1 or 2, wherein the histidine is histidine hydrochloride, and its concentration is: 0-100mmol / L, preferably 5-50mmol / L, more preferably 5-20mmol / L, and most preferably 10mmol / L; the arginine is arginine hydrochloride, and its concentration is: 0-160mmol / L, preferably 20-100mmol / L, more preferably 30-90mmol / L, and most preferably 35mmol / L. 4. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 3, wherein the glycerol content is: 0-1%, preferably 0.2-0.5% (w / v); and the mass percentage of Tween 80 is: 0-0.02% (w / v). 5. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 4, wherein the antibody in the antibody-drug conjugate is an anti-HER2 monoclonal antibody; and the drug conjugated to the antibody is MMAE or MMAF and its derivatives or DM1, DM4 and their derivatives. 6. The aqueous liquid pharmaceutical preparation according to item 5, wherein the anti-HER2 antibody drug conjugate is anti-HER2 monoclonal antibody-vc-MMAE, wherein the anti-HER2 monoclonal antibody and MMAE are connected via a linker vc, wherein the linker is combined with the free thiol groups exposed after reduction of the antibody via its thiol groups, wherein the structure after the linker and MMAE are connected is: 7. The aqueous liquid pharmaceutical formulation of item 5 or 6, wherein the anti-HER2 monoclonal antibody comprises a heavy chain and a light chain, (i) the heavy chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively; and / or (ii) the light chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively; The monoclonal antibody is preferably a chimeric antibody or a humanized antibody. 8. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 7, wherein the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5-30 mg / ml. 9. The aqueous liquid pharmaceutical preparation described in item 8, wherein the non-reducing sugar is 240-260 mmol / L mannitol and / or 40 mmol / L-60 mmol / L sucrose, the amino acid is 8-12 mmol / L histidine hydrochloride, and the solubilizing agent is 0-0.02% (w / v) Tween80. 10. The aqueous liquid pharmaceutical preparation of claim 9, wherein the non-reducing sugar is about 260 mmol / L mannitol and about 40 mmol / L sucrose, the amino acid is about 10 mmol / L histidine hydrochloride, the concentration of the antibody drug conjugate is about 10 mg / ml, and the solubilizing agent is 0.02% (w / v) Tween 80. 11. The aqueous liquid pharmaceutical preparation of claim 10, wherein the non-reducing sugar is about 240 mmol / L mannitol and about 60 mmol / L sucrose, the amino acid is about 10 mmol / L histidine hydrochloride, the concentration of the antibody drug conjugate is about 10 mg / ml, and the solubilizing agent is about 0.02% (w / v) Tween 80. 12. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 11, wherein the pH of the preparation is 4.5-7, preferably 5.6-6.8, more preferably 5.6-6.5, 5.6-6.4, 5.6-6.3, 6.1-6.4, 6.1-6.3. 13. A lyophilized pharmaceutical preparation of an antibody drug conjugate, the lyophilized pharmaceutical preparation being obtained by freeze-drying the aqueous liquid pharmaceutical preparation described in any one of items 1 to 12. 14. The lyophilized pharmaceutical preparation of item 13, wherein the aqueous liquid pharmaceutical preparation contains about 260 mmol / L of mannitol, about 40 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, about 0.02% (w / v) of Tween 80, about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and has a pH of 5.6-6.8. 15. The freeze-dried pharmaceutical preparation described in item 13, wherein the aqueous liquid pharmaceutical preparation contains about 240 mmol / L of mannitol, about 60 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, about 0.02% of Tween 80, about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and has a pH of 5.6-6.8. 16. The freeze-dried pharmaceutical preparation described in item 13, wherein the non-reducing sugars contained in the aqueous liquid pharmaceutical preparation are mannitol and sucrose, and the concentrations thereof are approximately 47.36 mg / ml and approximately 13.69 mg / ml, respectively; the amino acid is histidine hydrochloride, and the concentration thereof is approximately 2.10 mg / ml; and the solubilizing agent is Tween 80, and the content thereof is approximately 0.02% (w / v). 17. The freeze-dried pharmaceutical preparation described in item 13, wherein the non-reducing sugars contained in the aqueous liquid pharmaceutical preparation are mannitol and sucrose, and their concentrations are approximately 43.72 mg / ml and approximately 20.54 mg / ml, respectively; the amino acid is histidine hydrochloride, and its concentration is approximately 2.10 mg / ml; and the solubilizing agent is Tween 80, and its content is approximately 0.02% (w / v). 18. Use of the pharmaceutical preparation described in any one of items 1 to 17 in the preparation of a drug for treating a disease caused by abnormal expression of Her2, wherein the disease caused by abnormal expression is further preferably cancer; the cancer is further preferably a Her2-positive cancer; the Her2-positive cancer is further preferably breast cancer, ovarian cancer, gastric cancer, urothelial carcinoma, gastroesophageal cancer, esophageal cancer, endometrial cancer, lung cancer or bladder cancer. 19. A method for preparing an antibody-drug conjugate pharmaceutical preparation, comprising: (1) preparing a preparation of any one of items 1 to 17; (2) Evaluate the stability of the antibody drug conjugate in the formulation.

Claims

1. An aqueous liquid pharmaceutical preparation of an antibody drug conjugate, the preparation comprising an antibody drug conjugate, a non-reducing sugar, an amino acid, and a solubilizing agent; the non-reducing sugar is selected from mannitol, sucrose, trehalose or a combination thereof; the amino acid is selected from histidine, alanine, arginine, glycine, glutamic acid and the hydrochlorides of the above amino acids or a combination thereof; the solubilizing agent is selected from glycerol, Tween80 or a combination thereof.

2. The aqueous liquid pharmaceutical preparation of claim 1, wherein the mannitol concentration is 100-300 mmol / L, preferably 190-300 mmol / L, more preferably 200-260 mmol / L, and most preferably 240-260 mmol / L; the sucrose concentration is 0-100 mmol / L, preferably 40-100 mmol / L, more preferably 60-100 mmol / L, and most preferably 40-60 mmol / L.

3. The aqueous liquid pharmaceutical preparation according to claim 1 or 2, wherein the histidine is histidine hydrochloride, and its concentration is: 0-100mmol / L, preferably 5-50mmol / L, more preferably 5-20mmol / L, and most preferably 10mmol / L; the arginine is arginine hydrochloride, and its concentration is: 0-160mmol / L, preferably 20-100mmol / L, more preferably 30-90mmol / L, and most preferably 35mmol / L.

4. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 3, wherein the glycerol content is: 0-1%, preferably 0.2-0.5% (w / v); the mass percentage of Tween 80 is: 0-0.02% (w / v).

5. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 4, wherein the antibody in the antibody-drug conjugate is an anti-HER2 monoclonal antibody; and the drug conjugated to the antibody is MMAE or MMAF and its derivatives or DM1, DM4 and their derivatives.

6. The aqueous liquid pharmaceutical preparation according to claim 5, wherein the anti-HER2 antibody drug conjugate is anti-HER2 monoclonal antibody-vc-MMAE, wherein the anti-HER2 monoclonal antibody and MMAE are connected via a linker vc, wherein the linker is combined with the free thiol groups exposed after reduction of the antibody via its thiol groups, wherein the structure after the linker is connected to MMAE is:

7. The aqueous liquid pharmaceutical formulation of claim 5 or 6, wherein the anti-HER2 monoclonal antibody comprises a heavy chain and a light chain, (i) the heavy chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3, respectively; and / or (ii) the light chain comprises CDR1-3, wherein the amino acid sequences of CDR1-3 have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 6, respectively; The monoclonal antibody is preferably a chimeric antibody or a humanized antibody.

8. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 7, wherein the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5-30 mg / ml.

9. The aqueous liquid pharmaceutical preparation according to claim 8, wherein the non-reducing sugar is 240-260 mmol / L mannitol and / or 40 mmol / L-60 mmol / L sucrose, the amino acid is 8-12 mmol / L histidine hydrochloride, and the solubilizing agent is 0-0.02% (w / v) Tween 80 。 10. The aqueous liquid pharmaceutical formulation of claim 9, wherein the non-reducing sugar is about 260 mmol / L mannitol and about 40 mmol / L sucrose, the amino acid is about 10 mmol / L histidine hydrochloride, the concentration of the antibody drug conjugate is about 10 mg / ml, and the dissolution agent is 0.02% (w / v) Tween 80.

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