A pharmaceutical formulation comprising bevacizumab

Abstract

A pharmaceutical formulation containing 10-80 mg / ml bevacizumab is provided. The buffer system of the pharmaceutical formulation is preferably 10-30 mM histidine hydrochloride-sodium acetate buffer, contains 25-50 mg / mL sucrose or sorbitol as a stabilizer, and the surfactant Tween 80, with a pH of 5.0-5.6. The formulation of the present invention was tested under repeated freeze-thaw and accelerated conditions at 40°C, and the protein stability was good, which was superior to other similar formulation recipes.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a pharmaceutical formulation containing bevacizumab. Background Art

[0002] Highly specific, effective and safe protein (antibody) drugs, especially therapeutic antibody drugs, have become a hot spot in global drug research and development. Bevacizumab (also known as bevacumab, Chinese trade name: Anweiting, English trade name: Avastin) was developed by Roche and was first approved by the US FDA for marketing in 2004 and is widely used in the treatment of various malignant tumors, such as metastatic colorectal cancer, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, cervical cancer, glioblastoma, etc. Bevacizumab is a humanized monoclonal IgG1 antibody that can specifically bind to vascular endothelial growth factor, thereby blocking its binding to receptors (Flt-1 and KDR) on the surface of endothelial cells, avoiding a series of subsequent cascade reactions, inhibiting the formation of abnormal blood vessels, and then preventing tumor growth and spread, ultimately achieving the goal of eliminating tumors. In addition, bevacizumab has high specificity and usually does not interfere with other targets while blocking the VEGF pathway. Since bevacizumab can destroy abnormal blood vessels and normalize mature blood vessels, it is usually used in combination with chemotherapy, that is, acting on tumor tissues together with other drugs. In this treatment method, bevacizumab can effectively assist and consolidate the therapeutic effects of other drugs (Presta L G, Chen H, O'Connor S J, Chisholm V, Meng YG, Krummen L, et al. Cancer Res 1997; 57:4593-9.).

[0003] In the research of monoclonal antibody drugs, the research of pharmaceutical preparations plays an important role. IgG1 monoclonal antibodies mainly use injection liquid preparations, and proteins in liquid preparations are prone to form aggregates or particles, which affect stability. How to maintain good physical stability, chemical stability and biological stability of monoclonal antibody liquid preparations during the storage period has become a problem that cannot be ignored, and there is an urgent need to develop stable protein preparations that meet the requirements of the pharmaceutical industry. Summary of the Invention

[0004] The purpose of the present invention is to provide a pharmaceutical formulation containing bevacizumab.

[0005] In the present invention, through screening and formulation optimization, taking bevacizumab (HLX04 protein) as the research object, the buffer system was investigated; by designing single-factor experiments, the effects of different ionic strengths, pH values, types of stabilizers, types and contents of surfactants, etc. in the buffer system on protein stability were investigated under high-temperature acceleration conditions. And the ratio range of the contents of each component in the preparation was determined through experiments.

[0006] The detection items for evaluating the stability of the preparation protein under high-temperature accelerated conditions in the present invention include: appearance, protein concentration (A280), osmotic pressure, purity (SEC-HPLC, CEX-HPLC, CE-SDS), average protein particle size, PdI (DLS), and number of sub-visible particles (FlowCam).

[0007] The preferred pharmaceutical formulation of the present invention includes: bevacizumab, buffer, stabilizer, and surfactant; wherein the bevacizumab is a recombinant human monoclonal antibody, and its content is preferably 10 - 100 mg / mL, more preferably 10 - 80 mg / mL, or 10 - 50 mg / mL, and even more preferably 10 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL.

[0008] Preferably, the buffer in the present invention includes: one of histidine - histidine hydrochloride, acetic acid - sodium acetate, histidine hydrochloride - sodium acetate system; more preferably acetic acid - sodium acetate, histidine hydrochloride - sodium acetate buffer system; most preferably histidine hydrochloride - sodium acetate buffer system.

[0009] Among them, the pH value of the pharmaceutical formulation is preferably: pH 5.0 - 5.6, more preferably pH 5.3.

[0010] In this buffer system, the concentration of histidine - histidine hydrochloride, acetic acid - sodium acetate, and histidine hydrochloride - sodium acetate buffer is preferably 10 - 30 mM, among which, histidine hydrochloride - sodium acetate buffer is preferably 10 mM.

[0011] The preparation of the present invention also contains a stabilizer to protect the stability of the protein drug and protect the function of the protein drug from being affected by changes in conditions (such as freezing, lyophilization, or other preparation condition changes). The stabilizer is preferably selected from one or more of sucrose, trehalose, mannitol, sorbitol, or glycine; more preferably sucrose, trehalose, sorbitol, and even more preferably sucrose and sorbitol. The content of the stabilizer is preferably: 20 - 100 mg / mL; more preferably: 25 - 50 mg / mL; preferably: 45 mg / mL.

[0012] The surfactant is a conventional surfactant in the art, preferably a non-ionic surfactant. Examples of the surfactant in this article are preferably polysorbates. Among them, more preferably Tween 80. The content of the surfactant is preferably 0.1 - 0.5 mg / mL, more preferably 0.2 mg / mL.

[0013] The dosage form of the pharmaceutical formulation is a conventional dosage form in the art, preferably including liquid preparations for injection or freeze-dried preparations, etc. The liquid preparations for injection preferably include subcutaneous injection preparations, intravenous injection preparations, intraperitoneal administration preparations, intramuscular injection preparations, intravenous / subcutaneous injection preparations or intravitreal injection preparations, etc. The liquid preparations for injection preferably include aqueous injection preparations, prefilled syringe injection preparations, etc., and preferably an aqueous injection preparation, which can be used for intravenous injection or intravitreal injection.

[0014] Based on the results of single-factor studies, the preferred formulation prescription of bevacizumab was determined, and its composition was: 25 mg / mL bevacizumab, 10 mM histidine hydrochloride-sodium acetate, pH 5.3, 45 mg / mL sorbitol, 0.2 mg / mL Tween 80. According to the above ratio, the finished product was prepared, and accelerated stability studies and repeated freeze-thaw stability studies were carried out to verify the stability of the formulation of the present invention. And through stability test comparison with different formulation prescriptions, it can be seen from the analysis of the results of molecular isomers, charge isomers and subvisible particles in the high-temperature accelerated test that the stability of bevacizumab in the formulation of the present invention is significantly better than that of other prescriptions. Description of the Drawings

[0015] Figure 1 Investigation of the physicochemical properties of HLX04 protein in the buffer system of the prescription shown in Table 5;

[0016] Among them, (A) is the Tagg temperature at 0 week, (B) is the KD value at 0 week; (C) is the average particle size and polydispersity index PDI of the protein of HLX04 at 0 week and after standing at 40 °C for 4 weeks in the buffer system;

[0017] (D) is the percentage content of Pk 1Area of HLX04 protein at 0 week and after standing at 40 °C for 4 weeks in the buffer system; (E) the change trend of SEC aggregate content under the condition of 40 °C; (F) the change trend of CEX main peak content under the condition of 40 °C.

[0018] Figure 2 Investigation of the physicochemical properties of HLX04 protein in the buffer system of the prescription shown in Table 8;

[0019] Among them, (A) is a comparison diagram of thermodynamic stability, (B) the change trend of the average particle size of the protein under the condition of 40 °C; (C) the change trend of SEC aggregate content under the condition of 40 °C; (D) the change trend of CEX main peak content under the condition of 40 °C; (E) the change trend of the number of subvisible particles of FlowCam under the condition of 40 °C.

[0020] Figure 3 Investigation of the physicochemical properties of HLX04 protein in the buffer system of the prescription shown in Table 11;

[0021] Among them, (A) Thermodynamic stability comparison chart; (B) Trend of change in the average particle size of the protein at 40 °C; (C) Trend of change in the SEC aggregate content at 40 °C; (D) Trend of change in the CEX main peak content at 40 °C; (E) Trend of change in the number of subvisible particles detected by FlowCam at 40 °C.

[0022] Figure 4 Investigation of the physicochemical properties of HLX04 protein in the buffer system of the formulation shown in Table 14;

[0023] Among them, Figure (A) Thermodynamic stability comparison chart; (B) Trends of change in the average hydrodynamic diameter and PdI of DLS at 40 °C; (C) Trend of change in the SEC aggregate content at 40 °C; (D) Trend of change in the CEX main peak content at 40 °C; (E) Trend of change in the number of subvisible particles detected by FlowCam at 40 °C.

[0024] Figure 5H Investigation of the physicochemical properties of LX04 protein in the buffer system of the formulation shown in Table 20; Among them, (A) Thermodynamic stability comparison chart; (B) Trends of change in the average hydrodynamic diameter and PdI of DLS at 40 °C; (C) Trend of change in the SEC aggregate content at 40 °C; (D) Trend of change in the CEX main peak content at 40 °C; (E) Trend of change in the number of subvisible particles detected by FlowCam at 40 °C; (F) Trends of change in the average hydrodynamic diameter and PdI of DLS from -20 °C to room temperature; (G) Trend of change in the SEC aggregate content from -20 °C to room temperature; (H) Trend of change in the CEX main peak content from -20 °C to room temperature; (I) Trend of change in the number of subvisible particles detected by FlowCam from -20 °C to room temperature.

[0025] Figure 6 (A)(B)(C) Willingness characterizer model diagrams obtained by JMP software.

[0026] Figure 7 Investigation of the physicochemical properties of HLX04 protein in the buffer system of the formulation shown in Table 26; (A) Thermodynamic stability comparison chart; (B) Trend of change in the average protein particle size of DLS at 40 °C; (C)

[0027] Trend of change in the SEC main peak content at 40 °C; (D) Trend of change in the CEX main peak content at 40 °C; (E) Trend of change in the number of subvisible particles detected by FlowCam at 40 °C.

[0028] Figure 8 Stability comparison diagrams of different protein concentrations. (A) Trend of change in the SEC aggregate content; (B) Trend of change in the SEC fragment content; (C) Trend of change in the IgG content (CE-SDS non-reduced);

[0029] (D) Trend of change in the CEX main peak content. Detailed implementation methods

[0030] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art on how to implement and use the present invention, and do not limit the scope of the present invention. Nor does it mean that the experiments below are all the experiments that have been carried out and the only experiments that can be carried out.

[0031] All the chemical reagents used in the examples are commercially available analytical pure reagents. The recombinant monoclonal antibody can be a monoclonal antibody prepared by any currently known method. The following exemplary method for preparing antibodies is provided by Shanghai Fosun Pharma (Group) Co., Ltd., and this exemplary method does not limit the present invention.

[0032] The antibody protein used in this study is HLX04 (bevacizumab). Antibodies are prepared by the usual methods in the prior art, and their light and heavy chain sequences are as follows:

[0033] Light chain

[0034] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS

[0035] RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP

[0036] SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT

[0037] LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0038] Heavy chain

[0039] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPT

[0040] YAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL

[0041] VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV

[0042] LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL

[0043] LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0044] QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

[0045] REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0046] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0047] Example 1. Detection method

[0048] 1.1. Appearance and visible foreign matters

[0049] Visual inspection method was used for appearance detection. The sample vial was wiped clean and placed under the Shanghai Huanghai Pharmaceutical Inspection SC-4000A type clarity detector in a dark room. The illuminance was adjusted to 1000 - 1500 Lux. The sample was placed at the edge of the light-shielding plate (25 cm). Hold the neck of the test sample vial and visually inspect the color, clarity, and visible foreign matters against black and white backgrounds respectively.

[0050] 1.2. Protein content

[0051] Use a Trinean Dropsense16 type protein concentration detector to detect the absorbance of the sample at a wavelength of 280 nm and calculate the concentration. The extinction coefficient is 1.60 mL*mg -1 *cm -1 .

[0052] 1.3. pH value

[0053] Adopt a Mettler Toledo type multi-functional parameter instrument, calibrate with 3 standard solutions (pH values are 4.01, 7.00, and 9.21 respectively) to make the electrode slope within the range of 95% - 105%, and take 25 μL of the sample to measure the pH value.

[0054] 1.4. Osmolality

[0055] Using an AdvancedOsmoPRO osmometer, in accordance with the "Method for Determination of Osmolality" in General Chapter 0632 of the Chinese Pharmacopoeia (2015 Edition), take 20 μL of the sample and two portions of the osmotic pressure standard at 290 mOsmol / kg, and use the freezing point method to measure the osmotic pressure values of the sample and the standard.

[0056] 1.5. Viscosity

[0057] The viscosity of the sample was measured using a BROOKFIELDDV2T viscometer. Turn on the external water bath switch, set the temperature to 25 °C, suck 0.5 mL of the sample and drop it into the center of the sample cup, set the rotor speed so that the measurement torque falls within 40 - 60%, and measure the viscosity of the sample.

[0058] 1.6. DLS

[0059] The particle size and particle size distribution of the sample were measured using a DynaPro PlateReader-III high-throughput dynamic and static light scattering instrument. Take 25 μL of the sample in a clean bench and add it to the micro-wells of a 384-well plate. After adding the sample, cover the film, place the covered 384-well plate in a refrigerated centrifuge, and centrifuge to remove the air bubbles in the sample micro-wells. The specific instrument parameters are set as shown in Table 1.

[0060] Table 1 Parameter Settings for Particle Size Detection by High-Throughput Dynamic and Static Light Scattering Instrument

[0061]

[0062] 1.7. Tagg Temperature

[0063] The aggregation temperature of the sample was measured using a DynaPro PlateReader-III high-throughput dynamic and static light scattering instrument. Take 25 μL of the sample in a clean bench and add it to the micro-wells of a 384-well plate. After adding the sample, cover the film, place the covered 384-well plate in a refrigerated centrifuge, and centrifuge to remove the air bubbles in the sample micro-wells. The specific instrument parameters are set as shown in Table 2.

[0064] Table 2 Parameter Settings for Tagg Detection by High-Throughput Dynamic and Static Light Scattering Instrument

[0065]

[0066] 1.8. DSC

[0067] Using a TANano DSC differential scanning calorimeter, measure the thermodynamic parameters T monset , T m1 and T m2Value. Dilute the protein sample concentration to 1 mg / mL using placebo. Place the diluted protein sample and the corresponding placebo in a 96-well sample plate respectively. After degassing, place them under a pressure condition of 300 ± 50 Kpa. Set the pre-equilibration time to 600 s, the temperature range to 25 - 100 °C, and the scanning rate to 1 °C / min. Collect the DSC curves of the protein sample and the placebo respectively, with the placebo scanned three times and the protein sample scanned once. Select the Two State Scaled model for data fitting.

[0068] 1.9. SEC-HPLC

[0069] SEC-HPLC detection is performed using an Agilent 1260 high-performance liquid chromatography, with a TOSOH TSKgel G3000 chromatographic column (7.8 mm × 300 mm, 5 μm). The column temperature is room temperature (not temperature-controlled), the injection tray temperature is 2 - 8 °C, the mobile phase composition is 100 mM sodium dihydrogen phosphate, 0.5% sodium chloride, with a pH value of 6.8. Isocratic elution is carried out, the elution time is 30 min, and the flow rate is 0.5 mL / min. The detection wavelength is 280 nm, the sample concentration is diluted to 1 mg / mL, and the injection volume is 50 μL.

[0070] 1.10. CEX-HPLC

[0071] CEX-HPLC detection is performed using an Agilent 1260 high-performance liquid chromatography, ThermoProPac TM WCX-10 chromatographic column (4 mm × 250 mm, 10 μm). The column temperature is 35 °C, the injection tray temperature is 2 - 8 °C. The mobile phase A composition is 50 mM phosphate buffer with a pH value of 6.10, and the mobile phase B composition is 50 mM phosphate buffer, 300 mM sodium chloride, with a pH value of 6.10. Gradient elution is carried out, and the elution gradient is shown in Table 3. The flow rate is 1.0 mL / min. The detection wavelength is 280 nm, the sample concentration is diluted to 1 mg / mL, and the injection volume is 50 μL.

[0072] Table 3 CEX-HPLC elution gradient

[0073] Retention time Mobile phase B % Flow rate (mL / min) 0.0 min 4.0％ 1.0 3.0 min 4.0％ 1.0 31.0 min 31.0％ 1.0 32.0 min 100.0％ 1.0 34.0 min 100.0％ 1.0 34.1 min 4.0％ 1.0 39.0 min 4.0％ 1.0

[0074] 1.11. CE-SDS

[0075] Determined according to the general rule 3127 "Determination of Monoclonal Antibody Molecular Size Variation (CE-SDS Method)" in the Chinese Pharmacopoeia (2015 Edition). Non-reducing and reducing CE-SDS detections were adopted. Beckman Coulter PA800 plus capillary electrophoresis instrument was used, and an uncoated capillary with a total length of 67 cm and an inner diameter of 50 μm was used; injection was carried out at 5 KV for 20.0 s, separation was carried out at 15 KV for 35.0 min, a PDA detector was used, and detection was carried out at a wavelength of 220 nm; calculation was carried out by the area normalization method.

[0076] 1.12.FlowCam

[0077] The morphology and quantity of sub-visible particles in the sample were determined by using a FlowCam 8100 particle analysis detector. The specific parameter settings of the instrument are shown in Table 4.

[0078] Table 4 Detection Parameter Settings of FlowCam 8100 Particle Analysis Detector

[0079]

[0080] Example 2 Screening Research on Buffer System and Its pH Value

[0081] Regarding the research on buffer system / pH value, a total of two rounds of experiments were carried out, and the types of buffer systems and pH value ranges were screened through single-factor experimental design.

[0082] 2.1 Buffer System / pH Value Screening Research-I

[0083] 2.1.1 Research Plan

[0084] Table 5 Prescription Information of Buffer System / pH Value Screening Research-I

[0085]

[0086] In this research, the original HLX04 protein solution (batch number: AS201801) after removing Tween 20 by cation exchange chromatography was used. After ultrafiltration and buffer exchange and adjusting the protein concentration, an alternative prescription with a final protein concentration of about 25.0 mg / mL was prepared (Table 5). The sample was filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically aliquoted into 2 mL vials, with a 13 mm rubber stopper added and a 13 mm aluminum-plastic combination cap crimped. The aliquoted samples were placed in a constant temperature and humidity box at 40 °C for storage, and sampling and detection were carried out according to the design requirements in Table 6.

[0087] Table 6 Investigation Conditions and Detection Methods of Buffer System / pH Value Screening Research-I

[0088]

[0089] 2.1.1 Research Results

[0090] At week 0, in the histidine - histidine hydrochloride, acetic acid - sodium acetate, and histidine hydrochloride - sodium acetate systems, the Tagg temperature of the protein was relatively high ( Figure 1A ), the KD value was positive ( Figure 1B ), and the average particle size of the protein was relatively small ( Figure 1C ), indicating that the conformational stability and colloidal stability of the protein in these three buffer systems were relatively good.

[0091] After standing at 40 °C for 4 weeks, the results of protein concentration and pH value showed no obvious differences (Table 7). The SEC results showed that the main peak content of the protein decreased in each buffer system ( Figure 1E ). Among the three buffer systems of citric acid - sodium citrate, histidine - histidine hydrochloride, and acetic acid - sodium acetate, the lower the pH value, the better the stability. The ranking of superiority was C55 > A50 > H55 > HA55 > C60 ≈ A55 > H60 ( Figure 1E ). The DLS results showed that in the histidine - histidine hydrochloride and sodium dihydrogen phosphate - disodium hydrogen phosphate buffer systems, the dispersion index PdI increased ( Figure 1C ), and the percentage content of Pk 1Area Int decreased ( Figure 1D ), indicating the formation of soluble high polymers with a size below 1 μm. The CEX results showed that the main peak content of the protein decreased in each buffer system ( Figure 1F ), and the stability of the protein in the histidine - histidine hydrochloride buffer system was significantly better than that of other buffer systems.

[0092] Table 7 Summary of Data for Buffer System / pH Value Screening Study - I

[0093]

[0094] 2.2 Buffer System / pH Value Screening Study - II

[0095] 2.2.1 Research Plan

[0096] The results of the buffer system / pH value screening study - I showed that in the histidine - histidine hydrochloride, acetic acid - sodium acetate, and citric acid - sodium citrate buffer systems, the lower the pH value, the higher the SEC main peak content; in the formulations without stabilizers and surfactants, after being placed at 40 °C for 1 week, visible particles were observed in each formulation. Therefore, in this round of experiment, the buffer system was further screened in the formulations with a lower pH of 5.0 and the addition of sucrose and Tween 80.

[0097] Table 8 Formulation Information for Buffer System / pH Value Screening Study - II

[0098]

[0099] This study used the HLX04 protein (batch number: AS201901-PT). After ultrafiltration for buffer exchange, addition of excipients, and adjustment of protein concentration, an alternative formulation with a final protein concentration of approximately 25.0 mg / mL was prepared (Table 8). The samples were filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically aliquoted into 2 mL vials, capped with a 13 mm rubber stopper, and sealed with a 13 mm aluminum-plastic combination cap. The aliquoted samples were stored in a constant temperature and humidity chamber at 40 °C, and sampling and testing were performed according to the requirements of Table 9.

[0100] Table 9 Buffer system / pH value screening study - II Investigation conditions and detection methods

[0101]

[0102] 2.2.2 Research results

[0103] At week 0, the basic physicochemical test results of the protein in each buffer system showed no significant differences (Table 10). The order of the Tagg aggregation temperature from good to bad was A50≈HA50>H50>C50 ( Figure 2A ), and the order of the average particle size of the protein from good to bad was A50≈HA50≈H50>C50 ( Figure 2B ). This indicates that the protein has better conformational stability and colloidal stability in acetate-sodium acetate and histidine hydrochloride-sodium acetate buffer systems.

[0104] After standing at 40 °C for 4 weeks, the basic physicochemical test results showed no significant differences (Table 10). The SEC results showed that the main peak content of the protein decreased in each buffer system, and the order from good to bad was A50≈H50≈HA50>C50 ( Figure 2C ). The CEX results showed that the main peak content of the protein decreased in each buffer system, and the order from good to bad was H50≈HA50>A50>C50 ( Figure 2D ). The order of the subvisible particles detected by FlowCam from good to bad was A50>H50≈HA50>C50 ( Figure 2E ).

[0105] Table 10 Buffer system / pH value screening study - II Data summary

[0106]

[0107] 2.2.3 Research conclusions

[0108] The results of this round of research indicate that the protein has better conformational stability and colloidal stability in acetate-sodium acetate and histidine hydrochloride-sodium acetate buffer systems. The protein has better results for charge isomers in the histidine-hydrochloride histidine buffer system. Considering all factors, histidine hydrochloride-sodium acetate was selected as the buffer system for the protein.

[0109] Example 3 Screening of ionic strength

[0110] 3.1 Research plan

[0111] Table 11 Prescription information for ionic strength screening research

[0112]

[0113] In this study, HLX04 protein (batch number: AS201901-PT) was used. After ultrafiltration and buffer exchange, adding excipients, and adjusting the protein concentration, an alternative prescription with a final protein concentration of approximately 25.0 mg / mL was prepared (Table 11). The samples were filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically aliquoted into 2 mL vials, sealed with a 13 mm rubber stopper and crimped with a 13 mm aluminum-plastic combination cap. The aliquoted samples were stored in a constant temperature and humidity chamber at 40 °C, and sampling and testing were carried out according to the requirements designed in Table 12.

[0114] Table 12 Investigation conditions and detection methods for ionic strength screening research

[0115]

[0116] 3.2 Research results

[0117] At week 0, the order of the Tagg aggregation temperature ( Figure 3A ) and the average protein particle size ( Figure 3B ) of the protein in the histidine hydrochloride-sodium acetate system was HA-10 > HA-20 > HA-30, indicating that the conformational stability and colloidal stability of the protein were better in the 10 mM histidine hydrochloride-sodium acetate buffer system.

[0118] After standing at 40 °C for 4 weeks, the basic physical and chemical test results showed no significant differences (Table 13). The SEC results showed that the main peak content of the protein decreased in each buffer system ( Figure 3C ), and the order was HA-10 > HA-20 ≈ HA-30. The CEX results showed that the main peak content of the protein decreased in each buffer system, and the decreasing trend was not significantly different ( Figure 3D ). The order of sub-visible particles in FlowCam was HA-10 > HA-20 ≈ HA-30 ( Figure 3E ).

[0119] Table 13 Summary of ionic strength screening research data

[0120]

[0121] 3.3 Research conclusion

[0122] The results of this round of research indicate that the protein has good conformational stability and colloidal stability in the 10 mM histidine hydrochloride-sodium acetate buffer system. The content of the main peak in SEC shows a slow downward trend. There are fewer subvisible particles in FlowCam.

[0123] Screening of pH Value Range in Example 4

[0124] 4.1 Research Plan

[0125] Table 14 Prescription Information for pH Value Range Screening Research

[0126]

[0127] This research used HLX04 protein (batch number: AS201901-PT). After ultrafiltration and buffer exchange, adding excipients, and adjusting the protein concentration, an alternative prescription with a final protein concentration of approximately 25.0 mg / mL was prepared (Table 14). The samples were filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically aliquoted into 2 mL vials, sealed with a 13 mm rubber stopper, and capped with a 13 mm aluminum-plastic combination cap. The aliquoted samples were stored in a constant temperature and humidity chamber at 40 °C, and sampling and testing were carried out according to the requirements designed in Table 15.

[0128] Table 15 Investigation Conditions and Detection Methods for pH Value Range Screening Research

[0129]

[0130] 4.2 Research Results

[0131] The protein was stored in the 10 mM histidine hydrochloride-sodium acetate system for 4 weeks. There were no significant differences in the physicochemical properties of the protein within the pH range of 5.0 - 5.6 (Table 16, Figure 4). Therefore, the pH value range of the final formulation prescription is 5.0 - 5.6.

[0132] Table 16 Summary of Data for pH Value Range Screening Research

[0133]

[0134] Screening of Types of Stabilizers in Example 5

[0135] 5.1 Research Plan

[0136] Table 17 Prescription Information for Screening of Types of Stabilizers

[0137]

[0138] This research used HLX04 protein (batch number: AS201901-PT). After ultrafiltration and buffer exchange, adding excipients, and adjusting the protein concentration, an alternative prescription with a final protein concentration of approximately 25.0 mg / mL was prepared (表 17). Filter the sample with a 0.22 μm disposable sterile filter inside a biosafety cabinet, and then aseptically dispense 1 mL of the protein solution into 2 mL vials. Add a 13 mm rubber stopper and crimp a 13 mm aluminum-plastic combination cap. Examine and test the dispensed samples according to the requirements in Table 18.

[0139] Table 18 Investigation Conditions and Detection Methods for the Screening of Stabilizer Types

[0140]

[0141] 5.2 Research Results

[0142] At week 0, for the protein in the formulations containing various stabilizers, the order of temperature stability is sucrose > trehalose > sorbitol ≈ mannitol > glycine; the order of stability is glycine > mannitol ≈ sucrose ≈ trehalose ≈ sorbitol ( agg ). monset ). Figure 5A )

[0143] After standing at 40 °C for 4 weeks, the SEC results show that the main peak content of the protein in the formulations containing various stabilizers all shows a downward trend, and the downward trend of the glycine sample is significantly faster ( Figure 5C ). The order of DLS results is sorbitol ≈ mannitol > glycine > sucrose ≈ trehalose ( Figure 5B ). The CEX results show that the main peak content of the protein in the formulations containing various stabilizers all shows a downward trend, and the downward trend of the sample containing glycine is significantly faster ( Figure 5D ). The FlowCam results show that the order of subvisible particles is sorbitol ≈ sucrose ≈ trehalose ≈ mannitol > glycine ( Figure 5E )

[0144] After 10 rounds of repeated freeze-thaw cycles, the SEC results show that the main peak content of the protein in the formulations containing sucrose, trehalose, and sorbitol has not changed compared with week 0, and the downward trend of the main peak content in the formulations containing mannitol or glycine is significantly faster ( Figure 5G ). The order of DLS results is sorbitol > sucrose ≈ trehalose > glycine ≈ mannitol ( Figure 5F ). The CEX results show that the main peak content of the protein in the formulations containing sucrose, trehalose, and sorbitol has not changed compared with week 0, and the downward trend of the main peak content in the formulations containing mannitol and glycine is significantly faster ( Figure 5H ). The FlowCam results show that the order of subvisible particles is sorbitol ≈ sucrose ≈ trehalose > mannitol ≈ glycine ( Figure 5I and Table 19)

[0145] The results of this round of research show that under the conditions of standing at 40°C and repeated freezing and thawing, in the pH 5.3, 10 mM histidine hydrochloride-sodium acetate buffer system, the protein formulations with sucrose or sorbitol stabilizers have better stability.

[0146] Table 19 Summary of research data on the screening of stabilizer types

[0147]

[0148] Example 6 Screening of stabilizers and surfactants

[0149] 6.1 Research plan

[0150] In this round of research, using JMP 15 software, three factors including stabilizer type, stabilizer content, and Tween 80 content were selected, and 10 experimental groups were designed by the Box-Behnken response surface method (Table 20). The concentrations of stabilizers and surfactants were determined through accelerated conditions such as freezing and thawing, shaking, light, and high temperature (Table 21).

[0151] Table 20 Prescription information for the screening of stabilizers and surfactants

[0152]

[0153] This study used HLX04 protein (batch number: AS201901-PT), which was prepared into an alternative formulation with a final protein concentration of approximately 25.0 mg / mL after ultrafiltration and buffer exchange, addition of excipients, and adjustment of protein concentration (Table 20). The samples were filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically dispensed into 2 mL vials, with a 13 mm rubber stopper added and a 13 mm aluminum-plastic combination cap crimped. The dispensed samples were inspected and tested according to the requirements designed in Table 21.

[0154] Table 21 Investigation conditions and detection methods for the screening of stabilizers and surfactants

[0155]

[0156] 6.2 Research results

[0157] The detection data were imported into JMP 15 software, and the least squares method was used to perform multiple linear regression and binomial equation fitting on each factor (dependent variable) to obtain a model with statistical significance (P value < 0.1) (Table 22). Its adjusted determination coefficient (R2) was greater than 0.95, indicating that the model had a good fit with the actual situation, and the equation had good accuracy and reliability for the response value. This regression model could be used to analyze and predict the experimental results instead of the actual test points.

[0158] Using JMP 15 software, according to the analysis results in Table 22, a maximization willingness model was adopted to predict the optimal formulation prescription. When sorbitol was selected as the stabilizer, the willingness of the prescription was relatively high; when the sorbitol content was 4.5%, the willingness of the prescription was the highest; as the content of polysorbate 80 increased, the willingness of the prescription showed a downward trend. The willingness of the prescription was relatively high when the content of polysorbate 80 was in the range of 0.01 - 0.03%. The midpoint 0.02% was selected (see Figure 6A , 6B , 6C).

[0159] Through the buffer system / pH value screening study, ionic strength screening study, surfactant type screening study, stabilizer type screening study, and stabilizer and surfactant screening study, the final formulation prescription was as follows: 10 mM histidine hydrochloride-sodium acetate buffer system, pH 5.3, 45 mg / mL sorbitol, 0.2 mg / mL Tween 80.

[0160]

[0161] Comparison of the selected prescription in Example 7 with other prescriptions

[0162] 7.1 Research plan

[0163] Table 23 Prescription information

[0164]

[0165] In this study, HLX04 protein (batch number: AS201901-PT) was used. After ultrafiltration and buffer exchange, addition of excipients, and adjustment of protein concentration, alternative prescriptions with a final protein concentration of approximately 25.0 mg / mL were prepared (Table 23). Samples were filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 1 mL of the protein solution was aseptically aliquoted into 2 mL vials, with a 13 mm rubber stopper added and a 13 mm aluminum-plastic combination cap crimped on. The aliquoted samples were stored in a constant temperature and humidity chamber at 40 °C, and sampling and testing were carried out according to the design requirements in Table 24.

[0166] Table 24 Investigation conditions and detection methods

[0167]

[0168] 7.2 Research results

[0169] At week 0, the appearance of the protein in the three prescriptions was a colorless slightly opalescent liquid, without obvious visible foreign matters. The ranking of the Tagg aggregation temperature from good to bad was HA53 > PB62 > C50 ( Figure 7A ), and the ranking of the average protein particle size from good to bad was HA53 > C50 > PB62 ( Figure 7B ), indicating that the conformational stability and colloidal stability of the protein were better in the HA53 prescription.

[0170] After standing at 40 °C for 4 weeks, the protein could maintain a certain stability in all three formulations, and there were no obvious changes in the basic physical and chemical tests of the protein (Table 25). The SEC results showed that the main peak content of the protein showed a downward trend in each formulation ( Figure 7C ), and the ranking of superiority and inferiority was HA53 > C50 > PB62. The CEX results showed that the main peak content of the protein showed a downward trend in each formulation ( Figure 7D ), and the ranking of superiority and inferiority was HA53 > C50 > PB62. The ranking of superiority and inferiority of subvisible particles by FlowCam was HA53 > PB62 > C50 ( Figure 7E ).

[0171] Table 25 Summary of comparative research data for each formulation

[0172]

[0173] According to the results of molecular isomers, charge isomers and subvisible particles in the high-temperature accelerated test, the stability of bevacizumab in the formulations selected in this application was significantly better than that of the existing formulations (PB62) and other similar formulations used for bevacizumab.

[0174] Example 8 Comparison of stabilities at different protein concentrations

[0175] 8.1 Research plan

[0176] Table 26 Formulation information

[0177]

[0178] After the formulation screening study, the formulation of HLX04 preparation was determined as: 10 mM L-histidine hydrochloride-sodium acetate, 45 mg / mL sorbitol, 0.2 mg / mL Tween 80, pH 5.3. In this round of study, the high-temperature acceleration condition of 40 °C was adopted to compare the stability differences between samples in the concentration range of 10 - 80 mg / mL under the HLX04 preparation formulation and . HLX04 PT protein (batch number: AS201901-PT) was used, and after ultrafiltration and buffer exchange, addition of excipients, and adjustment of protein concentration, each alternative formulation was prepared (Table 26). The samples were filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 0.5 mL of the protein solution was aseptically aliquoted into 2 mL vials, with a 13 mm rubber stopper added and a 13 mm aluminum-plastic combination cap crimped. The aliquoted samples and the reference drug (batch number: H0154B14, code: Avastin) were placed in a constant temperature and humidity chamber at 40 °C for storage, and sampling and testing were carried out according to the design requirements in Table 27.

[0179] Table 27 Investigation conditions and detection methods

[0180]

[0181] 8.2 Research Results

[0182] In this study, through high-temperature (40°C) accelerated tests, the stability differences between samples within the concentration range of 10 - 80 mg / mL under the formulation of HLX04 and were compared. The research results showed (Table 28) that at week 0, the aggregate content of samples within the concentration range of 10 - 80 mg / mL under the formulation of HLX04 was 2.0 - 2.7%, less than that of Avastin (3.6%). After standing at 40°C for 4 weeks, the aggregation rate ( Figure 8A ) of samples within the concentration range of 10 - 80 mg / mL under the formulation of HLX04, the degradation rate ( Figure 8B , C), and the changing trend of charge isomers ( Figure 8D ) were all significantly slower than those of Avastin.

[0183] In summary, compared with the originator drug Avastin (60 mg / mL), the stability of the HLX04 formulation within the concentration range of 10 - 80 mg / mL is better.

[0184] Table 28 Summary of Stability Comparison Data at Different Protein Concentrations

[0185]

Claims

1. A pharmaceutical formulation comprising bevacizumab, characterized in that, The pharmaceutical formulation comprises: bevacizumab, a buffer, a stabilizer, and a surfactant; wherein: The protein concentration of the bevacizumab is 10 - 100 mg / mL; The buffer is a histidine hydrochloride - sodium acetate buffer, and its concentration is 10 - 30 mM; The stabilizer is sorbitol, and its content is 20 - 100 mg / mL; The surfactant is Tween 80 or Tween 20, and its content is 0.1 mg / mL - 0.5 mg / mL; Wherein the pH value of the pharmaceutical formulation is 5.0 - 5.

6.

2. The pharmaceutical formulation according to claim 1, characterized in that, The buffer is a histidine hydrochloride - sodium acetate buffer, and its concentration is 10 mM.

3. The pharmaceutical formulation according to claim 1, characterized in that, The protein concentration of the bevacizumab is 10 - 80 mg / mL.

4. The pharmaceutical formulation according to claim 3, characterized in that, The protein concentration of the bevacizumab is 10 - 50 mg / mL.

5. The pharmaceutical formulation according to any one of claims 1 to 3, characterized in that, The formulation contains: 10 mM histidine hydrochloride - sodium acetate buffer, 45 mg / mL sorbitol, 0.2 mg / mL Tween 80, pH 5.

3.

6. The pharmaceutical formulation according to claim 5, characterized in that The formulation contains 10 mg / mL, 25 mg / mL, 50 mg / mL, or 80 mg / mL bevacizumab.

7. The pharmaceutical formulation according to claim 1, characterized in that, The formulation is a liquid preparation for injection or a freeze - dried preparation.

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