Drug formulation containing bevacizumab

Abstract

Provided is a pharmaceutical formulation containing 10-80 mg / ml of bevacizumab, the buffer system of which is preferably 10-30 mM histidine hydrochloride-sodium acetate buffer, containing 25-50 mg / mL of sucrose or sorbitol as a stabilizer and Tween 80 as a surfactant, and having a pH of 5.0-5.6. The formulation has been tested under repeated freeze-thawing and high-temperature accelerated conditions at 40°C, and has good protein stability, superior to other similar formulations.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology, and specifically relates to a pharmaceutical preparation containing bevacizumab.

Background Art

[0002] Highly specific, effective and safe protein (antibody)-based drugs, especially therapeutic antibody drugs, have become a hot spot in current drug development worldwide. Bevacizumab (also known as Bevacizumab, Chinese trade name: Anvetin, English trade name: Avastin) was developed by Roche and was first approved by the US FDA and launched in 2004. It is widely applied to 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 endothelial cell surface, avoiding a subsequent series of cascade reactions, suppressing the formation of abnormal blood vessels, preventing the growth and spread of tumors, and ultimately achieving the goal of eliminating tumors. It should be noted that bevacizumab has strong specificity, blocks the VEGF pathway and usually does not interfere with other targets. Since bevacizumab can destroy abnormal blood vessels and normalize mature blood vessels, it is usually used in combination chemotherapy, that is, it acts on tumor tissues in combination with other drugs. In such a treatment method, bevacizumab can effectively assist and enhance the therapeutic effect 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 drugs, the research of drug formulations plays an important role. IgG1 monoclonal mainly adopts liquid formulations for injection. However, in liquid formulations, proteins are prone to form polymers or particles, which affect stability. How to maintain good physical, chemical, and biological stability during the storage period of monoclonal liquid formulations is an issue that cannot be ignored. The development of stable protein formulations that meet the needs of the pharmaceutical industry has become an urgent task.

Summary of the Invention

[0004] An object of the present invention is to provide a drug formulation containing bevacizumab.

[0005] In the present invention, through screening and formulation optimization, bevacizumab (HLX04 protein) is taken as the research object, the buffer system is considered, and single-factor experiments are designed to examine the effects of different ionic strengths, pH values, types of stabilizers, types of surfactants and their contents in the buffer system on protein stability under high-temperature accelerated conditions. Then, the mixing ratio ranges of the contents of each component in the formulation are determined through experiments.

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

[0007] A preferred drug formulation of the present invention contains bevacizumab, a buffer, a stabilizer, and a surfactant. Here, the bevacizumab is a recombinant human monoclonal antibody, and its content is desirably 10 - 100 mg / mL, preferably 10 - 80 mg / mL, or 10 - 50 mg / mL, and more preferably 10 mg / mL, 25 mg / mL, 50 mg / mL, 80 mg / mL.

[0008] Preferably, the buffer solution of the present invention contains one of histidine - histidine hydrochloride, acetic acid - sodium acetate, and histidine hydrochloride - sodium acetate systems. More preferably, it is an acetic acid - sodium acetate or histidine hydrochloride - sodium acetate buffer system. Most preferably, it is a histidine hydrochloride - sodium acetate buffer solution system.

[0009] Here, the pH value of the drug formulation is preferably pH 5.0 - 5.6, and more preferably pH 5.3.

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

[0011] The formulation of the present invention further contains a stabilizer to protect the stability of the protein drug and to 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, it is sucrose, trehalose, or sorbitol. Even more preferably, it is sucrose and sorbitol. The content of the stabilizer is preferably 20 - 100 mg / mL, more preferably 25 - 50 mg / mL, and most preferably 45 mg / mL.

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

[0013] The dosage form of the drug preparation is a conventional dosage form in the art, preferably including liquid preparations for injection or lyophilized preparations. The liquid preparation for injection preferably includes subcutaneous injection preparations, intravenous injection preparations, intraperitoneal administration preparations, intramuscular injection preparations, intravenous / subcutaneous injection preparations or intravitreal injection preparations. The liquid preparation for injection preferably includes liquid injection preparations, liquid prefilled injection preparations, etc., and is preferably a liquid injection preparation, and this liquid injection preparation may be used for intravenous injection or intravitreal injection.

[0014] Based on the results of single-factor studies, a suitable formulation of bevacizumab has been determined, and its composition is 25 mg / mL of bevacizumab, 10 mM of histidine hydrochloride-sodium acetate, pH 5.3, 45 mg / mL of sorbitol, and 0.2 mg / mL of Tween 80. Based on the above mixing ratio, the finished product is prepared, and the stability of the formulation of the present invention is verified by accelerated stability studies and repeated freeze-thaw stability studies. Then, through the comparison of stability tests with different formulations and the result analysis of molecular isomers, charge isomers and subvisible particles in the high-temperature accelerated test, it is found that the stability of bevacizumab in the formulation of the present invention is significantly better than that of other formulations.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0016] The following examples are used to provide those skilled in the art with a complete disclosure and description of how to implement and use the present invention, and do not limit the scope of the present invention. Nor do they mean that all the experiments below are all the experiments carried out and all the possible experiments.

[0017] All the chemical reagents used in the examples are commercially available analytical pure reagents. The recombinant monoclonal antibodies may be monoclonal antibodies prepared by any currently known method. The following exemplary antibody preparation method is provided by Shanghai Henlius Biotech, Inc., and this exemplary method does not limit the present invention.

[0018] The antibody protein used in this study is HLX04 (bevacizumab). Antibodies are prepared by the usual methods in the prior art, and the light and heavy chain sequences are as follows.

[0019] Light chain TIFF0007713950000001.tif25161

[0020] Heavy chain TIFF0007713950000002.tif51161

[0021] Example 1. Detection method 1.1. Appearance and visible foreign matters Visual inspection method is adopted for appearance detection. Wipe the sample vial clean, place it under the Shanghai Huanghai Pharmaceutical Inspection SC-4000A type transparency detector in a dark room, adjust the illuminance to 1000 - 1500 Lux, place the sample at the edge (25 cm) of the light shield, hold the neck of the test penicillin vial, and visually inspect the color, transparency and visible foreign matters, etc. against black and white backgrounds respectively.

[0022] 1.2. Protein content Using a Trinean Dropsense16 protein concentration meter, 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 is.

[0023] 1.3. pH value Adopt a Mettler Toledo multi-functional parameter meter, calibrate it with three standard solutions (the pH values are 4.01, 7.00, and 9.21 respectively), keep the electrode slope within the range of 95% - 105%, take 25 μL of the sample, and measure the pH value.

[0024] 1.4. Osmolality Adopt an Advanced Osmo PRO osmometer, measure according to the "Osmolality Measurement Method" in General Principles 0632 of the "Chinese Pharmacopoeia" (2015 Edition). Take 20 μL of the sample and two parts of an osmotic pressure standard of 290 mOsmol / kg, and measure the osmotic pressure values of the sample and the standard by the freezing point method.

[0025] 1.5. Viscosity Adopt a BROOKFIELD DV2T viscometer to measure the viscosity of the sample. Turn on the switch of the external water bath, 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 rotation speed so that the measured torque is 40% - 60%, and measure the viscosity of the sample.

[0026] 1.6. DLS Adopt a DynaPro PlateReader-III high-throughput dynamic light scattering meter to measure the particle size and particle size distribution of the sample. Take 25 μL of the sample in a clean bench and put it into the pores of a 384-orifice plate. After putting the sample, coat it. Put the coated 384-orifice plate into a refrigerated centrifuge and centrifuge to remove the air bubbles in the sample pores. The specific parameter settings of the instrument are as shown in Table 1.

[0027]

Table 1

[0028] 1.7. Tagg Temperature The DynaPro PlateReader-III high-throughput kinetic dynamic light scattering meter is used to measure the aggregation temperature of the sample. Take 25 μL of the sample in a clean bench and put it into the pores of a 384-well plate. After putting in the sample, cover it with a film. After covering the film, put the 384-well plate into a refrigerated centrifuge and centrifuge to remove the bubbles in the sample pores. The specific parameter settings of the instrument are as shown in Table 2.

[0029]

Table 2

[0030] 1.8. DSC The TA Nano DSC differential scanning calorimeter is used to measure the thermodynamic parameters T m onset , T m1 and T m2 values. Use placebo to dilute the protein sample concentration to 1 mg / mL. Put the diluted protein sample and the corresponding placebo into 96-well sample plates respectively. After degassing, place them under the pressure condition of 300 ± 50 Kpa. Set the pre-equilibration time to 600 s, the temperature range to 25 - 100 °C, and the scanning speed to 1 °C / min. Collect the DSC curves of the protein sample and the placebo respectively. Here, the placebo is scanned 3 times and the protein sample is scanned 1 time. Select the Two State Scaled model to perform data fitting.

[0031] 1.9. SEC-HPLC SEC-HPLC detection was performed using an Agilent 1260 high-performance liquid chromatography and a TOSOH TSKgel G3000 chromatographic column (7.8 mm × 300 mm, 5 μm). The column temperature was at room temperature (without temperature control), the temperature of the sample injection disk was 2 - 8°C, the mobile phase composition was 100 mM sodium dihydrogen phosphate and 0.5% sodium chloride, the pH value was 6.8, isocratic elution was carried out, the elution time was 30 min, and the flow rate was 0.5 mL / min. The detection wavelength was 280 nm, the sample concentration was diluted to 1 mg / mL, and the sample injection volume was 50 μL.

[0032] 1.10.CEX-HPLC CEX-HPLC detection was performed using an Agilent 1260 high-performance liquid chromatography and a Thermo ProPac TM WCX-10 chromatographic column (4 mm × 250 mm, 10 μm). The column temperature was 35°C, the temperature of the sample injection disk was 2 - 8°C, the composition of mobile phase A was 50 mM phosphate buffer with a pH value of 6.10, the composition of mobile phase B was 50 mM phosphate buffer and 300 mM sodium chloride with a pH value of 6.10, gradient elution was carried out, and the elution gradient was as shown in Table 3. The flow rate was 1.0 mL / min. The detection wavelength was 280 nm, the sample concentration was diluted to 1 mg / mL, and the sample injection volume was 50 μL.

[0033]

Table 3

[0034] 1.11.CE-SDS Measure in accordance with the general rule 3127 "Monoclonal Molecular Size Variation Measurement Method (CE - SDS Method)" in the Pharmacopoeia of the People's Republic of China (2015 Edition). Detect using non - reducing and reducing CE - SDS. Employ a Beckman Coulter PA800 plus type capillary electrophoresis apparatus. Use an uncoated capillary with a total length of 67 cm and an inner diameter of 50 μm. Inject the sample at 5 KV for 20.0 s, separate at 15 KV for 35.0 min, use a PDA detector, detect at a position where the wavelength is 220 nm, and calculate by the area normalization method.

[0035] 1.12.FlowCam Adopt a FlowCam 8100 particle analysis detector to measure the morphology and quantity of sub - visible particles in the sample. The specific parameter settings of the instrument are as shown in Table 4.

[0036]

Table 4

[0037] Example 2 Screening Research on Buffer Systems and Their pH Values For the research on buffer systems / pH values, a total of 2 experiments are conducted, and the types of buffer systems and pH value ranges are screened by the design of single - factor tests.

[0038] 2.1 Screening Research on Buffer Systems / pH Values - I 2.1.1 Research Plan

Table 5

[0039] This study uses the crude HLX04 protein solution (lot number: AS201801) after removing Tween 20 by cation chromatography. After performing liquid exchange by ultrafiltration and adjusting the protein concentration, it is prepared as an alternative formulation (Table 5) with a final protein concentration of approximately 25.0 mg / mL. Filter the sample using a 0.22 μm disposable sterile filter in a biosafety cabinet, and aseptically divide 1 mL of the protein solution into 2 mL penicillin bottles, add a 13 mm rubber stopper, and roll a 13 mm aluminum-plastic combination cap. Place the divided samples in a constant temperature and humidity box at 40°C for storage, and perform sampling and detection based on the design requirements in Table 6.

[0040]

Table 6

[0041] 2.1.1 Research Results At week 0, in the histidine - histidine hydrochloride, acetic acid - sodium acetate, and histidine hydrochloride - sodium acetate systems, the Tagg temperature of the protein is relatively high (Figure 1A), the KD value is a positive value (Figure 1B), and the average protein particle size is relatively small (Figure 1C), indicating that the conformational stability and colloidal stability of the protein in these three buffer systems are relatively good.

[0042] After standing at 40°C for 4 weeks, no significant difference was shown in the protein concentration and pH value results (Table 7). According to the SEC results, the main peak content of the protein in each buffer system showed a decreasing trend (Figure 1E). In the three buffer systems of citrate-sodium citrate, histidine-hydrochloric histidine, and acetic acid-sodium acetate, the lower the pH value, the relatively better the stability, and the ranking of superiority and inferiority was C55>A50>H55>HA55>C60≒A55>H60 (Figure 1E). According to the DLS results, the protein showed an increase in the dispersion index PdI (Figure 1C) and a decrease in the content rate of Pk 1 Area Int (Figure 1D) in the histidine-hydrochloric histidine and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer systems, indicating that soluble high polymers lower than 1 μm were generated. According to the CEX results, the main peak content of the protein in each buffer system showed a decreasing trend (Figure 1F), and it was shown that the stability of the protein in the histidine-hydrochloric histidine buffer system was significantly better than that of other buffer systems.

[0043]

Table 7

[0044] 2.2 Buffer system / pH value selection study - II 2.2.1 Research plan According to the results of the buffer system / pH value selection study - I, in the histidine-hydrochloric histidine, acetic acid-sodium acetate, and citrate-sodium citrate buffer systems, the lower the pH, the higher the SEC main peak content. In the formulation without stabilizer and surfactant, the protein was left at 40°C under the consideration conditions for 1 week, and it was shown that there were visible particles in each formulation. Therefore, in this experiment, the buffer system was further selected in the formulation with a relatively low pH of 5.0 and the addition of sucrose and Tween 80.

[0045]

Table 8

[0046] This study uses the HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, it is prepared as an alternative formulation (Table 8) with a final protein concentration of approximately 25.0 mg / mL. The samples are filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution is aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper is added, and a 13 mm aluminum-plastic combination lid is crimped. The divided samples are placed in a constant temperature and humidity box at 40 °C for storage, and sampling and detection are performed based on the design requirements in Table 9.

[0047]

Table 9

[0048] 2.2.2 Research Results At week 0, according to the basic physicochemical detection results of the protein in each buffer system, there was no significant difference (Table 10), and it was shown that the ranking of the Tagg aggregation temperature from high to low was A50 ≒ HA50 > H50 > C50 (Figure 2A), and the ranking of the average particle size of the protein was A50 ≒ HA50 ≒ H50 > C50 (Figure 2B). It was demonstrated that the conformational stability and colloidal stability of the protein in the acetic acid-sodium acetate and histidine hydrochloride-sodium acetate buffer systems were relatively good.

[0049] After standing at 40 °C for 4 weeks, the basic physicochemical detection results showed no significant difference (Table 10). According to the SEC results, the main peak content of proteins in each buffer system showed a decreasing trend, and the ranking was A50 ≒ H50 ≒ HA50 > C50 (Figure 2C). According to the CEX results, the main peak content of proteins in each buffer system showed a decreasing trend, and the ranking was H50 ≒ HA50 > A50 > C50 (Figure 2D). The ranking of FlowCam sub-visible particles was A50 > H50 ≒ HA50 > C50 (Figure 2E).

[0050]

Table 10

[0051] 2.2.3 Research conclusions The results of this study showed that the conformational stability and colloidal stability of proteins in the acetic acid-sodium acetate and histidine hydrochloride-sodium acetate buffer systems were relatively good. The results of the charge isomers of proteins in the histidine-hydrochloric acid histidine buffer system were relatively good. Considering comprehensively, histidine hydrochloride-sodium acetate was selected as the buffer system for proteins.

[0052] Example 3 Selection of ionic strength 3.1 Research plan

Table 11

[0053] This study uses the HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, it is prepared as an alternative formulation (Table 11) with a final protein concentration of approximately 25.0 mg / mL. The samples are filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution is aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper is added, and a 13 mm aluminum-plastic combination lid is crimped. The divided samples are placed in a constant temperature and humidity box at 40 °C for storage, and sampling and detection are performed based on the design requirements in Table 12.

[0054]

Table 12

[0055] 3.2 Research Results At week 0, the ranking 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 in the 10 mM histidine hydrochloride-sodium acetate buffer system were relatively good.

[0056] After standing at 40 °C for 4 weeks, the basic physicochemical detection results showed no significant difference (Table 13). According to the SEC results, the main peak content of the protein in each buffer system showed a decreasing trend (Figure 3C), and the ranking was HA-10 > HA-20 ≒ HA-30. According to the CEX results, the main peak content of the protein in each buffer system showed a decreasing trend, and there was no significant difference in the decreasing trend (Figure 3D). The ranking of the FlowCam sub-visible particles was HA-10 > HA-20 ≒ HA-30 (Figure 3E).

[0057]

Table 13

[0058] 3.3 Research Conclusions According to the results of this study, it was shown that the conformational stability and colloidal stability of proteins in a 10 mM histidine hydrochloride-sodium acetate buffer system were relatively good. The decreasing trend of the SEC main peak content was relatively slow. The FlowCam subvisible particles were relatively few.

[0059] Example 4 Selection of pH Value Range 4.1 Research Plan [Table 14]

[0060] This study adopted the HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, it was prepared as an alternative formulation (Table 14) with a final protein concentration of approximately 25.0 mg / mL. The samples were filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution was aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper was added, and a 13 mm aluminum-plastic combination lid was rolled. The divided samples were placed in a constant temperature and humidity box at 40 °C for storage, and sampling and detection were performed based on the design requirements in Table 15.

[0061] [Table 15]

[0062] 4.2 Research Results The protein was stored in a 10 mM histidine hydrochloride-sodium acetate system for 4 weeks, and there was no significant difference 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 is 5.0 - 5.6.

[0063]

Table 16

[0064] Example 5 Selection of Stabilizer Types 5.1 Research Plan

Table 17

[0065] This study uses the HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, it is prepared as an alternative formulation (Table 17) with a final protein concentration of approximately 25.0 mg / mL. The sample is filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution is aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper is added, and a 13 mm aluminum-plastic combination cap is crimped. For the divided samples, considerations and detections are carried out based on the design requirements in Table 18.

[0066]

Table 18

[0067] 5.2 Research Results At week 0, for the protein in each formulation containing stabilizer, the temperature ranking from best to worst is sucrose > trehalose > sorbitol ≈ mannitol > glycine, and the stability ranking from best to worst is glycine > mannitol ≈ sucrose ≈ trehalose ≈ sorbitol (Figure 5A). agg m onset

[0068] ​​After standing at 40 °C for 4 weeks, according to the SEC results, the main peak content of the protein in each formulation containing a stabilizer showed a decreasing trend, and it was shown that the decreasing trend of the glycine sample was obvious and relatively fast (Figure 5C). The ranking of the DLS results is sorbitol ≈ mannitol > glycine > sucrose ≈ trehalose (Figure 5B). According to the CEX results, the main peak content of the protein in each formulation containing a stabilizer showed a decreasing trend, and it was shown that the decreasing trend of the glycine-containing sample was obvious and relatively fast (Figure 5D). According to the FlowCam results, it was shown that the ranking of the subvisible particles was sorbitol ≈ sucrose ≈ trehalose ≈ mannitol > glycine (Figure 5E).

[0069] After 10 cycles of repeated freeze-thaw, according to the SEC results, the main peak content of the protein in the formulations containing sucrose, trehalose, and sorbitol did not change compared with that at 0 week, and it was shown that the decreasing trend of the main peak content in the formulations containing mannitol or glycine was obvious and relatively fast (Figure 5G). The ranking of the DLS results is sorbitol > sucrose ≈ trehalose > glycine ≈ mannitol (Figure 5F). According to the CEX results, the main peak content of the protein in the formulations containing sucrose, trehalose, and sorbitol did not change compared with that at 0 week, and it was shown that the decreasing trend of the main peak content in the formulations containing mannitol and glycine was obvious and relatively fast (Figure 5H). According to the FlowCam results, it was shown that the ranking of the subvisible particles was sorbitol ≈ sucrose ≈ trehalose > mannitol ≈ glycine (Figure 5I and Table 19).

[0070] According to the results of this study, under the conditions of high-temperature standing at 40 °C and repeated freeze-thaw, in a 10 mM histidine hydrochloride-sodium acetate buffer system with a pH of 5.3, the formulations added with sucrose or sorbitol stabilizers showed relatively good protein stability.

[0071]

Table 19

[0072] Example 6 Selection of Stabilizer and Surfactant 6.1 Research Plan This study used JMP 15 software to select three factors: stabilizer type, stabilizer content, and Tween 80 content, and adopted the Box-Behnken response surface method to design 10 test groups (Table 20), and determined the concentrations of stabilizer and surfactant (Table 21) under accelerated conditions such as freeze-thaw, shaking, light irradiation, and high temperature.

[0073]

Table 20

[0074] This study adopted HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, an alternative formulation with a final protein concentration of approximately 25.0 mg / mL was obtained. In this study, JMP software was used to select three factors: stabilizer type, stabilizer content, and the content of Tween 80, and the Box-Behnken response surface method was adopted to design 10 test groups (Table 20), and the concentrations of stabilizer and surfactant (Table 21) were determined under accelerated conditions such as freeze-thaw, shaking, light irradiation, and high temperature. The samples were filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution was aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper was added, and a 13 mm aluminum-plastic combination cap was rolled. For the divided samples, considerations and detections were carried out based on the design requirements of the table.

[0075]

Table 21

[0076] 6.2 Research Results The detection data was introduced into JMP 15 software, and multiple linear regression and binomial equation fitting were performed for each element (dependent variable) using the least squares method. A statistically significant (P-value < 0.1) model (Table 22) was obtained, and its adjusted determination coefficient (R2) was greater than 0.95 in all cases. The fitting between the model and the actual situation was good, and the equation had relatively good accuracy and reliability for the response value. It was demonstrated that instead of the actual test points, this regression model could be used to analyze and predict the experimental results.

[0077] Apply JMP 15 software and predict the optimal formulation based on the analysis results in Table 22 using the maximum satisfaction model. When sorbitol is selected as the stabilizer, the satisfaction of the formulation is relatively high. When the sorbitol content is 4.5%, the formulation satisfaction is the highest. As the content of polysorbate 80 increases, the formulation satisfaction shows a downward trend. When the content of polysorbate 80 is within the range of 0.01 - 0.03%, the formulation satisfaction is relatively large, and the midpoint 0.02% is selected (see Figures 6A, 6B, 6C).

[0078] 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 composition is 10 mM histidine hydrochloride - sodium acetate buffer system, pH 5.3, 45 mg / mL sorbitol, and 0.2 mg / mL Tween 80.

[0079]

Table 22

[0080] Example 7 Comparison between the Selected Formulation and Other Formulations 7.1 Research Plan

Table 23

[0081] This study uses the HLX04 protein (lot number: AS201901-PT). After performing liquid exchange by ultrafiltration, adding auxiliary materials, and adjusting the protein concentration, it is prepared as an alternative formulation (Table 23) with a final protein concentration of approximately 25.0 mg / mL. The samples are filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and 1 mL of the protein solution is aseptically divided into 2 mL penicillin bottles, a 13 mm rubber stopper is added, and a 13 mm aluminum-plastic combination cap is crimped. The divided samples are placed in a constant temperature and humidity box at 40°C for storage, and sampling and detection are performed based on the design requirements in Table 24.

[0082]

Table 24

[0083] 7.2 Research Results At week 0, the protein in all three formulations is a colorless liquid with slight opalescence and no obvious visible foreign matter. The ranking of the Tagg aggregation temperature from high to low is HA53 > PB62 > C50 (Figure 7A), and the ranking of the average protein particle size from high to low is HA53 > C50 > PB62 (Figure 7B), indicating that the conformational stability and colloidal stability of the protein in the HA53 formulation are relatively good.

[0084] After standing at 40°C for 4 weeks, the protein in all three formulations can maintain a certain stability, and there are no significant changes in the basic physicochemical detection of the protein (Table 25). According to the SEC results, the main peak content of the protein in each formulation shows a decreasing trend (Figure 7C), and the ranking from high to low is HA53 > C50 > PB62. According to the CEX results, the main peak content of the protein in each formulation shows a decreasing trend (Figure 7D), and the ranking from high to low is HA53 > C50 > PB62. The ranking of the FlowCam sub-visible particles from high to low is HA53 > PB62 > C50 (Figure 7E).

[0085]

Table 25

[0086] According to the results of molecular isomers, charge isomers and subvisible particles in the high-temperature accelerated test, the stability of bevacizumab in the formulation selected in this application is significantly better than that of the formulation (PB62) used in conventional bevacizumab and other similar formulations.

[0087] Example 8 Comparison of Stability at Different Protein Concentrations 8.1 Research Plan

Table 26

[0088] Through the formulation screening study, it was determined that the HLX04 formulation is 10 mM histidine hydrochloride-sodium acetate, 45 mg / mL sorbitol, 0.2 mg / mL Tween 80, and pH 5.3. In this study, high-temperature accelerated conditions of 40 °C were adopted, and in the HLX04 formulation, the stability differences between samples in the concentration range of 10 - 80 mg / mL and Avastin (registered trademark) were compared. HLX04 PT protein (lot number: AS201901-PT) was adopted. After liquid exchange by ultrafiltration, addition of auxiliary materials, and adjustment of protein concentration, it was prepared as each alternative formulation (Table 26). Samples were filtered using a 0.22 μm disposable sterile filter in a biosafety cabinet, and then 0.5 mL of the protein solution was aseptically dispensed into 2 mL penicillin bottles, a 13 mm rubber stopper was added, and a 13 mm aluminum-plastic combination lid was crimped. The separated samples and the reference drug (lot number: H0154B14, code: Avastin) were placed in a constant temperature and humidity box at 40 °C for storage, and sampling and detection were carried out based on the design requirements in Table 27.

[0089]

Table 27

[0090] 8.2 Research Results This study compares the stability differences between samples in the concentration range of 10 - 80 mg / mL and Avastin (registered trademark) in the HLX04 formulation by means of a high-temperature (40°C) accelerated test. According to the research results (Table 28), at week 0, in the HLX04 formulation, the polymer content of samples in the concentration range of 10 - 80 mg / mL was shown to be 2.0 - 2.7%, which is less than that of Avastin (3.6%). After standing at 40°C for 4 weeks, in the HLX04 formulation, the aggregation rate (Figure 8A), degradation rate (Figures 8B, C), and charge isomer change tendency (Figure 8D) of samples in the concentration range of 10 - 80 mg / mL were all significantly slower than those of Avastin.

[0091] As described above, compared with the reference drug Avastin (60 mg / mL), the HLX04 formulation in the concentration range of 10 - 80 mg / mL has better stability.

[0092]

Table 28

Claims

1. A pharmaceutical preparation containing bevacizumab, wherein the pharmaceutical preparation contains bevacizumab, a buffer, a stabilizer, and a surfactant, the buffer is a histidine hydrochloride-sodium acetate buffer with a concentration of 10-30 mM, the stabilizer is sorbitol with a content of 20-100 mg / mL, the surfactant is Tween 80 or Tween 20 with a content of 0.1 mg / mL - 0.5 mg / mL, and the pH value of the pharmaceutical preparation is 5.0 - 5.

6. A pharmaceutical preparation containing bevacizumab, characterized by this.

2. The pharmaceutical preparation according to Claim 1, characterized in that the concentration of the histidine hydrochloride-sodium acetate buffer is 10 mM.

3. The pharmaceutical preparation according to Claim 1, characterized in that the surfactant is Tween 80 with a content of 0.1 mg / mL - 0.5 mg / mL.

4. The protein concentration of the bevacizumab is 10-100 mg / mL, preferably 10-80 mg / mL, or the protein concentration of the bevacizumab is 10-50 mg / mL. The pharmaceutical preparation according to Claim 1, characterized by this.

5. The pharmaceutical preparation according to any one of Claims 1 to 4, characterized in that the preparation contains a 10 mM histidine hydrochloride-sodium acetate buffer, 45 mg / mL of sorbitol, and 0.2 mg / mL of Tween 80 and has a pH of 5.

3.

6. The pharmaceutical preparation according to Claim 5, characterized in that the preparation contains 10 mg / mL, 25 mg / mL, 50 mg / mL, or 80 mg / mL of bevacizumab.

7. The pharmaceutical preparation according to Claim 1, characterized in that it is a liquid preparation for injection or a lyophilized preparation.

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