Anti-her2 monoclonal antibody lyophilized formulation and method of preparation

Abstract

The application relates to a stable freeze-dried preparation of anti-HER2 monoclonal antibody and a preparation method, wherein the defects of protein solution instability are greatly improved by optimizing the preparation formula and the freeze-drying process, the medicine produced by the freeze-dried preparation formula and the preparation method has a 25 DEG C stability of at least 6 months and a 5 DEG C stability of at least 60 months.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to a lyophilized formulation of an anti-HER2 monoclonal antibody and its preparation method. Background Technology

[0002] HER2 / neu (human epidermal growth factor receptor 2), also known as erbB2, is a member of the human epidermal growth factor receptor family and possesses tyrosine protein kinase activity. It is expressed at low levels only in a few normal tissues of adults. However, studies have shown that HER2 is overexpressed in various tumors, such as in approximately 30% of breast cancer patients and 16% of gastric cancer patients. HER2 overexpression in tumors can significantly promote tumor angiogenesis, tumor growth, and enhance tumor invasion and metastasis, making it an important indicator of poor prognosis in these patients.

[0003] PCT patent application WO2020 / 025013 discloses a self-developed recombinant anti-HER2 humanized monoclonal antibody, 19H6-Hu, which specifically binds to domain DIII of the extracellular domain (HER2-ECD) of human HER2. It is a recombinant humanized monoclonal antibody expressed in CHO cells using DNA recombination technology, derived from the humanization of the heavy and light chain variable regions of an anti-HER2 mouse antibody. In-depth research on 19H6-Hu revealed numerous charge isomers in its protein solution, which tend to increase during storage; therefore, formulation optimization is needed to improve stability. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to provide a lyophilized formulation of an anti-HER2 monoclonal antibody and its preparation method. The lyophilized formulation comprises an anti-HER2 monoclonal antibody, a buffer solution, a protein protectant, and a surfactant. The preparation method includes steps such as drug solution aliquoting and freeze-drying.

[0005] To achieve the objectives of this invention, the following technical solution is adopted:

[0006] The first aspect of the present invention provides a lyophilized formulation of an anti-HER2 monoclonal antibody, the lyophilized formulation comprising an anti-HER2 monoclonal antibody, a buffer, a protein protectant, and a surfactant.

[0007] The concentration of the anti-HER2 monoclonal antibody is 30-80 mg / ml. The concentration of the anti-HER2 monoclonal antibody can be any of the following ranges: 30-40 mg / ml, 40-50 mg / ml, 50-60 mg / ml, 60-70 mg / ml, or 70-80 mg / ml.

[0008] The anti-HER2 monoclonal antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2. Preferably, the concentration of the anti-HER2 monoclonal antibody is 40 mg / ml.

[0009] The buffer solution is a histidine-histidine hydrochloride buffer or a phosphate buffer. The histidine hydrochloride is histidine hydrochloride. The concentration of the buffer solution is 10-20 mM. The concentration of the buffer solution can be any of the following ranges: 10-12 mM, 12-14 mM, 14-16 mM, 16-18 mM, or 18-20 mM. Preferably, the buffer solution is a histidine-histidine hydrochloride buffer. The concentration of the buffer solution is 10 mM. The concentration of the buffer solution refers to the sum of the molar concentrations of histidine and histidine hydrochloride contained in the buffer solution.

[0010] The protein protectant is trehalose or sucrose. The concentration of the protein protectant is 30-40 mg / ml. The concentration of the protein protectant can be any of the following ranges: 30-32 mg / ml, 32-34 mg / ml, 34-36 mg / ml, 36-38 mg / ml, or 38-40 mg / ml. Preferably, the protein protectant is trehalose. The concentration of the protein protectant is 30 mg / ml.

[0011] The surfactant is polysorbate 80. The concentration of the surfactant is 0.01-1.0 mg / ml. The concentration of the surfactant can be any of the following ranges: 0.01-0.1 mg / ml, 0.1-0.3 mg / ml, 0.3-0.5 mg / ml, 0.5-0.7 mg / ml, or 0.7-1.0 mg / ml. Preferably, the concentration of the surfactant is 0.3 mg / ml.

[0012] The lyophilized formulation has a pH of 5.2-7.0. Preferably, the lyophilized formulation has a pH of 5.2-6.3. The pH range of the lyophilized formulation is any of the following: 5.2-5.4, 5.4-5.6, 5.6-5.7, 5.7-5.8, 5.8-6.0, 6.0-6.2, 6.2-6.3, 6.3-6.4, 6.4-6.6, 6.6-6.8, or 6.8-7.0. More preferably, the lyophilized formulation has a pH of 5.7-6.3.

[0013] A second aspect of the present invention provides a method for preparing the aforementioned lyophilized formulation, the method comprising the steps of dispensing the drug solution and freeze-drying.

[0014] The dosage of the drug solution dispensed is 30-420 mg / bottle. Preferably, the dosage of the drug solution dispensed is 210-420 mg / bottle. More preferably, the dosage of the drug solution dispensed is 210 mg / bottle.

[0015] The freeze-drying process includes pre-freezing, primary drying, secondary drying, and final drying steps.

[0016] The pre-freezing temperature is 5℃, the pre-freezing temperature is -40℃, and the pre-freezing time is 180-185 minutes. The main drying temperature is -10 to -20℃, the time is 17-30 hours, and the vacuum degree is 200-300 μbar. The secondary drying temperature is 0-15℃, the time is 5-15 hours, and the vacuum degree is 100-300 μbar. The final drying temperature is 30℃, the time is 2.5-4 hours, and the vacuum degree is 20-60 μbar.

[0017] Preferably, the pre-freezing temperature is 5°C, the pre-freezing temperature is -40°C, and the pre-freezing time is 185 min. The main drying temperature is -15°C, the time is 26 h, and the vacuum degree is 300 μbar. The secondary drying temperature is 0-15°C, the time is 740 min, and the vacuum degree is 200-300 μbar. The final drying temperature is 30°C, the time is 2.5 h, and the vacuum degree is 20-60 μbar.

[0018] A third aspect of the invention provides the use of the described lyophilized formulation in the preparation of a medicament for treating cancer.

[0019] The cancers mentioned include breast cancer, stomach cancer, or ovarian cancer.

[0020] When the lyophilized anti-HER2 monoclonal antibody formulation of this invention is administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined by referring to the results of animal experiments and various other factors, but the total dosage should not exceed a certain range.

[0021] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0022] A fourth aspect of the present invention provides a method for treating cancer, the method comprising administering the lyophilized formulation to a subject in need.

[0023] The cancers mentioned include breast cancer, stomach cancer, or ovarian cancer.

[0024] When the lyophilized formulation is administered to a subject, the dosage must be a therapeutically effective amount. The therapeutically effective amount refers to an amount that is effective in treating cancer. Specifically, when the lyophilized formulation is administered to a subject, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration, and may be based on animal experiment results and various other factors; the total dosage must not exceed a certain range.

[0025] Beneficial effects: This invention provides a stable lyophilized formulation of an anti-HER2 monoclonal antibody and its preparation method. This invention significantly improves upon the instability of protein solutions by optimizing the formulation and lyophilization process. Drugs produced according to the lyophilized formulation and preparation method of this invention exhibit stability of at least 6 months at 25°C and at least 60 months at 5°C. Attached Figure Description

[0026] Figure 1 The effects of pH, excipients, and dosage form on protein purity were analyzed.

[0027] Figure 2 For DOE result analysis.

[0028] Figure 3 The results of the investigation of buffer solutions and protein protectants.

[0029] Figure 4 The freeze-drying curve is shown in Figure 4-1.

[0030] Figure 5 The freeze-drying curve is shown in Figure 4-2.

[0031] Figure 6 The freeze-drying curve is shown in section 4-3.

[0032] Figure 7 The change in IEC purity is shown at 5℃. Detailed Implementation

[0033] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0035] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0036] The protein samples used in the following examples are derived from the anti-HER2 monoclonal antibody 19H6-Hu disclosed in WO2020 / 025013, and its heavy chain and light chain amino acid sequences are shown below.

[0037] The amino acid sequence of the 19H6-Hu heavy chain (SEQ ID NO: 1)

[0038]

[0039] The amino acid sequence of the 19H6-Hu light chain (SEQ ID NO: 2)

[0040]

[0041] The detection methods used in the following embodiments are described below:

[0042] SEC purity and polymer detection methods:

[0043] Mobile phase: 200 mM phosphate buffer, pH 6.8 ± 0.1. Filtered through a 0.22 μm filter and degassed by sonication before use. Column: TSK G3000SWxl, 7.8 × 300 mm 5 μm, TOSOH 08541. High-performance liquid chromatograph: Waters Alliance e2695 2489 UV / Vis detector, Dionex Ultimate 3000 VWD-3400(RS) Detector, or other suitable HPLC system equipped with a UV detector.

[0044] System suitability sample: Dilute the reference sample to 5.0 mg / ml with the mobile phase, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a sample vial, and place it in the HPLC sample tray. Test sample: Dilute the test sample to 5.0 mg / ml with the mobile phase, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a sample vial, and place it in the HPLC sample tray. Chromatographic conditions: Column temperature 25±2℃; Sample temperature 10±2℃; Detection wavelength UV 280nm; Injection volume 20 μL; Flow rate 0.5 ml / min.

[0045] Integrating data using chromatographic software, the peak area percentage of each peak was calculated using the peak area normalization method. System suitability acceptance criteria: For 6-needle system suitability samples, the resolution between polymers and monomers is ≥1.5, the retention time RSD of the main peak is ≤1.0%, the peak area RSD of the main peak is ≤2.0%, and the asymmetry of the main peak is ≤2.0%, with a theoretical plate number ≥4000 for all samples. Test sample report results: The SEC purity report for the sample is the percentage of the peak area of ​​the monomer main peak, and the polymer content is the percentage of the peak area of ​​the polymer peak.

[0046] IEC purity testing methods:

[0047] Mobile phase A: 20 mM phosphate buffer, pH 6.5 ± 0.05. Filtered through a 0.22 μm filter and degassed by sonication before use. Mobile phase B: 20 ​​mM phosphate buffer + 200 mM sodium chloride, pH 6.5 ± 0.05. Filtered through a 0.22 μm filter and degassed by sonication before use. Column: Propac WCX-10, 4 × 250 mm, Thermo Dionex 054993. High-performance liquid chromatograph: Waters Alliance e2695, Dionex Ultimate 3000 series, or other suitable HPLC system equipped with a UV detector.

[0048] System suitability sample: Dilute the reference sample to 1.0 mg / ml with the mobile phase, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a sample vial, and place it in the HPLC sample tray. Test sample: Dilute the test sample to 1.0 mg / ml with the mobile phase, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a sample vial, and place it in the HPLC sample tray. Chromatographic conditions: Column temperature 30±2℃; Sample temperature 10±2℃; Detection wavelength UV 214nm; Injection volume 20 μL; Flow rate 1.0 ml / min. Mobile phase gradient is as follows:

[0049]

[0050] Purity analysis: The peak area percentages of the main peak, acid peak region, and basic peak region on the sample spectrum are calculated using the peak area normalization method. IEC purity results are reported as the percentage of the main peak's peak area.

[0051] Unless otherwise specified, all raw materials used in the following examples are commercially available.

[0052] Example 1: Effects of pH, excipients, and dosage form on protein purity

[0053] This example investigated the effects of different pH ranges (4.0-7.0), different buffer types (20mM acetate buffer, 20mM phosphate buffer, 20mM histidine-histidine hydrochloride buffer), different protein concentrations (30-80 mg / ml), different polysorbate 80 concentrations (0.2-2.0 mg / ml), different protein protectants (sucrose, trehalose), and different dosage forms (injection, lyophilized) on the purity of the protein SEC and IEC. Ten formulations were prepared according to Table 1. The injection volume was 1 ml with a dose of 30 mg, and the lyophilized volume was 5.3 ml with a dose of 420 mg. The lyophilization parameters are shown in Table 2. The samples were stored at 40℃ and tested for SEC and IEC purity at 0, 3, 5, 7, and 10 days.

[0054] Table 1. Effects of pH, excipients, and dosage form on protein purity

[0055]

[0056] Table 2, 1-9, 1-10 Freeze-drying parameters used in the freeze-drying group

[0057] Put into box 5 \ \ Pre-freezing -40 \ 5 Pre-freezing -40 \ 180 dry -20 250 40 dry -20 250 540 dry -10 250 25 dry -10 250 120 dry 10 250 40

[0058] dry 30 60 20 dry 30 60 220

[0059] Table 3. Results of experiments on the effects of pH, excipients, and dosage form on protein purity.

[0060]

[0061] From Table 3 and Figure 1The results showed that the SEC purity and IEC purity of groups 1-9 and 1-10 were significantly better than those of other groups, indicating that the lyophilized formulations were more stable than the injectable solutions. From the IEC purity results, except for the lyophilized group, groups 1-4, 1-5, 1-6, 1-7, and 1-8 were better than groups 1-1, 1-2, and 1-3, indicating that lower pH values ​​are detrimental to IEC purity. The differences between groups 1-1 and 1-2 were small, indicating that different polysorbate 80 contents had little effect, and all values ​​from 0.2 to 2.0 mg / ml met the requirements. The differences between groups 1-8 and 1-7 are not significant, indicating that the concentration of protein at 80 mg / ml is not significantly different from that at 30 mg / ml. Dispensing 1.0-5.3 ml volumes into 2-20 ml vials can cover doses of 30-420 mg / vial. The differences between groups 1-4, 1-5, 1-6, and 1-7 are not significant, indicating that there is no significant difference between histidine-histidine hydrochloride buffer and phosphate buffer, which is superior to acetate buffer. The differences between groups 1-9 and 1-10 are not significant, indicating that there is no significant difference between sucrose and trehalose.

[0062] Example 2: DOE investigation of pH and surfactant

[0063] This embodiment uses a Design of Effect (DOE) design to investigate pH and polysorbate 80 dosage. The pH range is set to 5.2-6.2, and the polysorbate 80 content range is set to 0.01-1.0 mg / ml. The protein content is 40 mg / ml, the histidine-histidine hydrochloride buffer is 20 mM, and the trehalose dosage is 40 mg / ml. The filling volume is 1 ml, and the dosage is 40 mg / bottle (solution). Ten formulations were incubated at 40°C for 4 weeks, and samples were taken at 0, 1, 2, 3, and 4 weeks to test SEC purity, IEC purity, and insoluble particulates.

[0064] Table 4. DOE Design Recipe Table

[0065]

[0066] Table 5. DOE Results

[0067]

[0068]

[0069]

[0070] Linear regression analysis was performed on the DOE results for each group in Table 5. The slope of each group was analyzed using the JMP DOE model, and the results are as follows. Figure 2 As shown. By Figure 2As can be seen, the shaded area in the figure represents the optimal screening range. The preferred pH range is 5.2-6.2, and the preferred dosage of polysorbate 80 is 0.01-1.0 mg / ml. Specifically, when the pH range is higher, the dosage of polysorbate 80 is lower, and when the pH range is lower, the dosage of polysorbate 80 is higher.

[0071] Example 3: Investigation of buffer concentration and protein protectant concentration

[0072] This embodiment investigated the concentrations of the buffer solution and the protein protectant. As shown in Table 6, five batches of samples were prepared. The histidine-histidine hydrochloride buffer solution ranged from 5-20 mM, the trehalose concentration was 20-40 mg / ml, and the pH was 6.0. After sample preparation, the samples were lyophilized at a volume of 5.3 ml and a dosage of 210 mg / vial. The lyophilization parameters are shown in Table 7. After lyophilization, the samples were stored at 40°C, and samples were taken at 0, 1, 2, 3, and 4 weeks to determine the purity of SEC and IEC.

[0073] Table 6. Investigation of buffer concentration and protein protectant concentration

[0074]

[0075]

[0076] Table 7. Lyophilization parameters for test buffer and protein protectant

[0077] Put into box 5 \ \ Pre-freezing -40 \ 5 Pre-freezing -40 \ 180 dry -20 300 40 dry -20 300 960 dry 0 300 120 dry 0 300 360 dry 15 100 40 dry 15 100 120 dry 30 60 20 dry 30 60 130

[0078] Table 8. Results of buffer and protein protectant treatments

[0079]

[0080]

[0081] From Table 8 and Figure 3 The results show that group 3-1 had poor purity in both SEC and IEC, while group 3-4 had poor SEC purity. Group 3-2 had the best IEC purity, and group 3-5 had the best SEC purity. Based on these results, the optimal buffer concentration is 10-20 mM, and the optimal trehalose concentration is 30-40 mg / ml.

[0082] Example 4: Investigation of the freeze-drying process

[0083] The lyophilization process was investigated according to the parameters in Table 9. The lyophilization formulation was: protein 40 mg / ml, histidine-histidine hydrochloride buffer 10 mM, trehalose 30 mg / ml, polysorbate 80 0.3 mg / ml, pH 6.0. 5.3 ml of the solution was filled into 20 ml vials, with a dosage of 210 mg / vial. The lyophilization curve and sample appearance were then examined.

[0084] Table 9. Investigation of Freeze-drying Process

[0085]

[0086] Depend on Figures 4-6 The results show that the main drying time of the 4-1 freeze-drying curve was insufficient, and the product curve did not intersect with the plate temperature line, which may have prevented the water from sublimating completely. Curves 4-2 and 4-3, however, met the requirements. The earliest intersection of curve 4-2 was at approximately 1020 min (17 h), indicating that the main drying time was at least 17 h. The appearance of the samples after three freeze-drying cycles all met the requirements. Therefore, the preferred freeze-drying parameters are as follows: 5°C initial temperature, pre-freezing at -40°C for 3 h; main drying temperature -15 to -20°C, vacuum degree 200 to 300 μbar, time ≥17 h; secondary drying 0 to 15°C, vacuum degree 100 to 300 μbar, time 5 to 10 h; final drying 30°C, vacuum degree 60 μbar, time 4 h.

[0087] Example 5 Stability Study

[0088] As shown in Table 10, a batch of pharmaceutical products was prepared according to the preferred formulation of this invention: 40 mg / ml protein, 10 mM histidine-histidine hydrochloride, 30 mg / ml trehalose, 0.3 mg / ml polysorbate 80, and pH 6.0 ± 0.3. The product was filled to a volume of 5.3 ml, with a dosage of 210 mg / vial. Stability was assessed after lyophilization at 25°C and 5°C, with SEC purity and IEC purity being tested.

[0089] Table 10. Stability Batch Freeze-Drying Parameters

[0090] Put into box 5 \ \ Pre-freezing -40 \ 5 Pre-freezing -40 \ 180 Empty \ 300 \ dry -15 300 60 dry -15 300 1500 dry 0 300 60 dry 0 300 480 dry 15 200 20 dry 15 200 180 dry 30 60 20 dry 30 60 60 dry 30 20 10 dry 30 20 60

[0091] Table 11. Stability Results

[0092]

[0093]

[0094] As shown in Table 11, after being stored at 25℃ for 6 months, the changes in SEC purity and IEC purity were minimal, meeting the quality standard requirements. (Table 11 and...) Figure 7The results showed that after being stored at 5°C for 6 months, the purity of SEC did not change significantly, and the shelf life of the drug calculated according to ICH guidelines based on IEC purity was 91 months. Therefore, the drug produced according to the formulation and preparation method of this invention has a stability that supports a shelf life of 60 months.

[0095] Example 6: Stability Study of Pilot-Scale Samples

[0096] Three pilot-scale samples were produced according to the preferred formulation of this invention: protein 40 mg / ml, histidine-histidine hydrochloride 10 mM, trehalose 30 mg / ml, polysorbate 80 0.3 mg / ml, pH 6.0±0.3. The batch size was 2000-4000 vials, the filling volume was 5.3 ml, and the dosage was 210 mg / vial. The lyophilization parameters were consistent with those in Table 10. After lyophilization, accelerated stability was assessed at 25±2℃ and long-term stability was assessed at 5±3℃. The tests included SEC purity and IEC purity, and the results are shown in Table 12.

[0097] Table 12. Stability results of pilot-scale samples

[0098]

[0099]

[0100] As shown in Table 12, the SEC purity and IEC purity of each batch of samples did not change significantly within 12 months of long-term stability, and the accelerated stability trend was similar to that of the batch studied in Example 5. Therefore, the drug has good stability.

[0101] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention. sequence list <110> 3SBio (Shanghai) Co., Ltd. <120> Lyophilized formulation and preparation method of anti-HER2 monoclonal antibody <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 447 <212> PRT <210> Artificial Sequence <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Ile His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Ile 35 40 45 Gly Val Phe Ser Ile Tyr Tyr Glu Asn Ile Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Met Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Gly Gly Thr Ile Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 2 <211> 219 <212> PRT <210> Artificial Sequence <400> 2 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Ile Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A lyophilized formulation of an anti-HER2 monoclonal antibody, characterized in that, The lyophilized formulation comprises: an anti-HER2 monoclonal antibody, a buffer solution, a protein protectant, and a surfactant; wherein the anti-HER2 monoclonal antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2; the concentration of the anti-HER2 monoclonal antibody is 40 mg / ml; the buffer solution is histidine-histidine hydrochloride buffer, and the concentration of the buffer solution is 10 mM; the protein protectant is trehalose, and the concentration of the protein protectant is 30 mg / ml; the surfactant is polysorbate 80, and the concentration of the surfactant is 0.3 mg / ml; the pH of the lyophilized formulation is 5.7-6.

3. The lyophilized formulation is prepared as follows: including the steps of dispensing the drug solution and freeze-drying; the dosage of the drug solution dispensed is 210 mg / bottle; The freeze-drying process includes pre-freezing, main drying, secondary drying, and final drying steps. The pre-freezing inlet temperature is 5°C, the pre-freezing temperature is -40°C, and the pre-freezing time is 180-185 min. The main drying temperature is -10 to -20°C, the time is 17-30 h, and the vacuum degree is 200-300 μbar. The secondary drying temperature is 0-15°C, the time is 5-15 h, and the vacuum degree is 100-300 μbar. The final drying temperature is 30°C, the time is 2.5-4 h, and the vacuum degree is 20-60 μbar.

2. The lyophilized formulation according to claim 1, characterized in that, The pre-freezing temperature is 5℃, the pre-freezing temperature is -40℃, and the pre-freezing time is 185 min; the main drying temperature is -15℃, the time is 26 h, and the vacuum degree is 300 μbar; the secondary drying temperature is 0-15℃, the time is 740 min, and the vacuum degree is 200-300 μbar; the final drying temperature is 30℃, the time is 2.5 h, and the vacuum degree is 20-60 μbar.

3. Use of the lyophilized formulation as described in claim 1 in the preparation of a medicament for treating cancer; The cancers mentioned are selected from the following group: breast cancer, stomach cancer, and ovarian cancer.

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