CN111375057B - Pharmaceutical formulation comprising anti-Her 2 monoclonal antibody - Google Patents

Pharmaceutical formulation comprising anti-Her 2 monoclonal antibody Download PDF

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CN111375057B
CN111375057B CN201811628635.0A CN201811628635A CN111375057B CN 111375057 B CN111375057 B CN 111375057B CN 201811628635 A CN201811628635 A CN 201811628635A CN 111375057 B CN111375057 B CN 111375057B
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hlx11
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monoclonal antibody
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CN111375057A (en
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马崇军
韩冬梅
姜伟东
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Shanghai Fuhong Hanlin Biopharmacy Co ltd
Shanghai Henlius Biotech Inc
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Shanghai Henlius Biotech Inc
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Abstract

The present invention provides a pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody, the pharmaceutical formulation comprising: anti-Her 2 monoclonal antibody, buffer solution, isotonic regulator, protein protectant, surfactant, etc. The product is stored for a certain time under the room temperature condition or higher temperature, and the detection shows that the main detection items have no obvious change, and all detection results meet the current quality standard requirements of the product. The pharmaceutical formulation obtained by the invention can ensure the validity period of more than four weeks under the storage condition of not higher than 50 ℃. Compared with the original ground pharmaceutical preparation, the pharmaceutical preparation has better stability and is more beneficial to the stability of the antibody.

Description

Pharmaceutical formulation comprising anti-Her 2 monoclonal antibody
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a pharmaceutical preparation containing an anti-Her 2 monoclonal antibody.
Background
Pertuzumab (trade name: perjeta) is a recombinant humanized monoclonal antibody that targets HER 2. The drug was first approved by the us FDA for marketing in 2012 for use in combination with trastuzumab and docetaxel for treatment of Her2 positive metastatic breast cancer patients without prior anti-Her 2 treatment or chemotherapeutic treatment history of metastatic disease.
Protein (monoclonal antibody etc.) pharmaceutical formulations generally include active ingredients, buffer ingredients, isotonic regulators, stabilizers and the like. The saccharides or alcohols are common auxiliary materials added into the medicines, mainly play a role of a stabilizer, have the characteristics of good stability, no accumulation of human bodies, safety and the like, and can help the biological macromolecular medicines to maintain the molecular structure and the biological activity of the biological macromolecular medicines, thereby being beneficial to the long-term stability of the medicines. Sodium chloride is used as an isotonic regulator, and a small amount of sodium chloride is added into the protein solution to increase the electric charge on the surface of protein molecules and enhance the action of the protein molecules and water molecules, so that the solubility of the protein in the aqueous solution is increased. The surfactant is a common pharmaceutical adjuvant in antibody pharmaceutical preparations, and can reduce the surface tension of gas-liquid and solid-liquid interfaces, thereby reducing aggregation of proteins at two-phase interfaces, and being beneficial to maintaining the stability in the processes of production, transportation and the like.
In the research process of protein (monoclonal antibody and the like) pharmaceutical preparations, detection technologies related to the stability of the drugs comprise basic physicochemical property detection, protein purity detection, particle detection, protein activity detection and the like. The basic physicochemical property detection items comprise appearance, pH value, protein concentration, osmolality and the like, the protein purity detection includes molecular isomer (SEC-HPLC, SDS-PAGE) detection, charge isomer (CEX-HPLC, cIEF) detection and the like, and the particle detection generally uses imaging and counting Flowcam of sub-visible particles, dynamic light scattering DLS, a photoresistance method and the like.
The protein (monoclonal antibody, etc.) pharmaceutical formulations are suitable for parenteral administration, including intravenous, intramuscular, intraperitoneal, or subcutaneous injection. The tendency of proteins in liquid formulations to form aggregates or particles has been a problem in protein pharmaceutical formulations. In general pharmaceutical formulations, proteins, particularly monoclonal antibodies, are included which are difficult to maintain in good physical, chemical and biological stability during storage.
Disclosure of Invention
In order to provide protein formulations with good stability meeting the demands of the pharmaceutical industry, the present invention provides a new pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody. Through high-throughput screening and formula optimization research, the protein preparation with higher stability is obtained.
The buffer system was screened to examine its effect on protein stability. And (3) designing a single factor test, and examining the influence of different ionic strength, protein concentration, pH value, stabilizer types and surfactant types on protein stability.
In order to solve the technical problems, one of the technical schemes adopted by the invention is as follows: a pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody, the pharmaceutical formulation comprising: anti-Her 2 monoclonal antibodies, buffers, protein protectants, and surfactants.
Wherein the "pharmaceutical formulation" is a conventional pharmaceutical formulation in the art, generally refers to a preparation in a form that allows for the biological activity of the active ingredient to be effective, the subject's suitability to meet clinical needs and not cause adverse reactions, such formulation being sterile. The pharmaceutical formulation may also include pharmaceutically acceptable excipients (vehicles or additives) which are those adjuvant components that can be reasonably administered to the subject mammal to provide an effective dose.
The dosage form of the pharmaceutical formulation is conventional in the art, and preferably includes a liquid preparation for injection or a lyophilized preparation, etc. The liquid preparation for injection preferably includes a subcutaneous injection preparation, an intravenous injection preparation, an intraperitoneal administration preparation, an intramuscular injection preparation, an intravenous/subcutaneous injection preparation, a vitreous injection preparation, or the like. The liquid preparation for injection preferably includes a water needle injection preparation, a prefilled needle injection preparation, and the like, preferably a water needle injection preparation, which can be used for intravenous injection.
Wherein the anti-Her 2 monoclonal antibody is a conventional anti-Her 2 monoclonal antibody in the field, more preferably a recombinant anti-Her 2 fully human monoclonal antibody, and the anti-Her 2 monoclonal antibody is preferably Pertuzumab (Pertuzumab), preferably the HLX11 monoclonal antibody prepared according to the invention. The light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 1 in the sequence table, and the heavy chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO.2 in the sequence table. The content of the anti-Her 2 monoclonal antibody is preferably 5mg/ml to 50mg/ml, more preferably 10mg/ml to 40mg/ml, and preferably 30mg/ml.
The preparation method of the anti-Her 2 monoclonal antibody is a conventional preparation method in the field, and the preparation method is briefly described as follows: engineering cell lines were constructed by transfecting CHO cells with recombinant plasmids encoding recombinant anti-Her 2 domain II humanized monoclonal antibody genes.
The production cell culture process comprises seed cell resuscitating, shake flask amplifying, WAVE bioreactor amplifying and one-time reactor production cell culturing, wherein the production cell culturing is carried out by adopting a basal medium containing HM004 and a feed medium containing HF 001. During the culture process, glutamine, a feed medium, glucose and sodium butyrate are supplemented. Culture conditions: viable cell density after inoculation was 0.7X10 6±0.1×106 cells/mL; stirring at 90rpm; pH 6.90+ -0.10; dissolved oxygen is saturated concentration initially, and then is stabilized near the set 40%; the initial temperature is 37.0 ℃; adding sodium butyrate for 6-8 hours, and cooling to 33.0 ℃. Harvested after 15 days of culture or when the cell activity is below 60.0%.
The protein production and purification process flow comprises deep filtration, affinity chromatography, low pH virus inactivation, anion chromatography, cation chromatography, virus removal filtration, ultrafiltration concentration and liquid exchange, the finally obtained protein product is prepared into a table 1 prescription, the table 1 prescription is placed in a constant temperature shaking table, and is shaken for 2 weeks under the conditions of 40 ℃ and 200rpm, sampling is carried out at the 0 th week, the 1 st week and the 2 nd week respectively, and detection and analysis are carried out, wherein the detection items comprise protein content (A 280), molecular isomer (SEC-HPLC) and charge isomer (CEX-HPLC).
Wherein the buffer is conventional in the art, "buffer" generally refers to a buffer solution that resists changes in pH by the action of its acid-base complex components. Preferably, the buffer according to the present invention comprises: one of buffer systems such as citric acid-sodium citrate, histidine-histidine hydrochloride, acetic acid-sodium acetate or citric acid-disodium hydrogen phosphate; more preferably a citric acid-sodium citrate or histidine-histidine hydrochloride buffer system; most preferred is a histidine-hcl buffer system. The concentration of the buffer is preferably: 10-30mM, more preferably 15-25mM, preferably 20mM.
The preparation method of the anti-Her 2 monoclonal antibody can also refer to the disclosures of Stancovski et al PNAS (USA) 88:8691-8695 (1991)) and Fendly et al cancer Research 50:1550-1558 (1990), and the preparation method of the anti-Her 2 monoclonal antibody is the technical content known to the person skilled in the art.
Wherein the protein protectant is a conventional protein protectant in the art, or referred to as a "protein stabilizer". The protein protectant is capable of protecting the stability of the protein drug and protecting the function of the protein drug from changes in conditions (e.g., freezing, lyophilization or other changes in manufacturing conditions). The protein protectant preferably comprises a saccharide, protein, amino acid, polymer, salt, amine, surfactant, etc., more preferably one or more of sorbitol, sucrose, trehalose, mannitol, arginine hydrochloride, or glycine; preferably sorbitol. The mass percentage content of the protein protectant is preferably as follows: 1% -5%, more preferably 2% -4%, preferably 3%.
Wherein the surfactant is a surfactant conventional in the art, preferably a nonionic surfactant. Examples of surfactants herein preferably include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearoyl-sulfobetaines; lauryl-, myristyl-, linoleyl-, or stearoyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocoamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g., lauramidopropyl betaine); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl cocoyl taurate; MONAQUATTM series (Mona Industries Inc, paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., pluronics, PF 68), and the like. The surfactant is more preferably polysorbate 20 and/or polysorbate 80, preferably polysorbate 20. The surfactant is preferably present in an amount of 0.1mg/mL-0.4mg/mL, more preferably 0.15mg/mL-0.35mg/mL, and preferably 0.2mg/mL.
The pH of the pharmaceutical formulation of the invention is conventional in the art, preferably: pH5-7, more preferably: pH5.5-6.5, preferably: pH6.0.
The pharmaceutical formulation of the present invention may optionally contain an isotonic regulator, which is conventional in the art, preferably glucose or sodium chloride, etc., more preferably sodium chloride.
The pharmaceutical formulation of the present invention preferably comprises: 30mg/mL of anti-Her 2 monoclonal antibody, 20mmol/L of histidine-histidine hydrochloride buffer, 3% sorbitol by mass, 0.2mg/mL of polysorbate 20, and pH of 6.0.
The pharmaceutical formulations of the present invention are in the form of conventional dosage forms in the art. Preferably, the composition comprises a liquid preparation, a solid preparation, a freeze-dried preparation and the like; more preferably, the pharmaceutical formulation is a liquid formulation for injection or a lyophilized formulation.
The medicine preparation prepared by the invention is stored for a certain time under the condition of room temperature or higher (40-50 ℃), all main detection indexes have no obvious change, and the medicine preparation has better stability than the original preparation. The pharmaceutical formulation containing the anti-Her 2 monoclonal antibody better solves the problem of stability of protein drugs, and can ensure the validity period of more than four weeks under the storage condition of not higher than 50 ℃.
Drawings
Fig. 1: HLX11 pharmaceutical formulation high throughput buffer system screening for 2 week purity change comparison results. Wherein, fig. 1 (a): SEC main peak content variation versus plots for different prescriptions; fig. 1 (B): CEX main peak content variation versus plots for different formulations.
Fig. 2: HLX11 pharmaceutical formulation buffer system screening 4 week purity change comparison results. Wherein, fig. 2 (a): SEC main peak content variation versus plots for different prescriptions; fig. 2 (B): SEC fragment content variation versus plots for different prescriptions; fig. 2 (C): CEX main peak content variation contrast plots for different prescriptions; fig. 2 (D): CEX acid peak content variation plots for different formulations; fig. 2 (E): comparison of mean particle size variation of DLS proteins for different formulations.
Fig. 3: HLX11 pharmaceutical formulation adjuvant screening 4 week purity change comparison results. Wherein, fig. 3 (a): SEC main peak content change trend comparison graphs of different prescriptions; fig. 3 (B): a SEC fragment content change trend comparison chart of different prescriptions; fig. 3 (C): CEX main peak content change trend comparison diagrams of different prescriptions; fig. 3 (D): CEX acidic peak content change trend comparison graphs of different prescriptions; fig. 3 (E): comparison of mean particle size variation of DLS proteins for different formulations.
Fig. 4: HLX11 formulation surfactant screening 4 week purity change comparison results. Wherein, fig. 4 (a): SEC main peak content variation versus plots for different prescriptions; fig. 4 (B): SEC fragment content variation versus plots for different prescriptions; fig. 4 (C): CEX main peak content variation contrast plots for different prescriptions; fig. 4 (D): CEX acid peak content variation versus plots for different formulations.
Fig. 5: HLX11 drug formulation preliminary stability studies accelerate the results of the variation in the content of each component of SEC and CEX. Fig. 5 (a): HLX11 drug formulation and SEC main peak content trend profile for Perjeta; fig. 5 (B): HLX11 drug formulation and SEC fragment content trend graph of Perjeta; fig. 5 (C): linear fit trend plots of the changes in SEC main peak content for HLX11 drug formulation and Perjeta; fig. 5 (D): trend graphs were linearly fitted for HLX11 drug formulation and SEC fragment content changes of Perjeta. Fig. 5 (E): HLX11 drug formulation and CEX main peak content trend plot of Perjeta; fig. 5 (F): HLX11 drug formulation and CEX acid peak content trend plot of Perjeta; fig. 5 (G): linear fit trend plots of the changes in CEX main peak content for HLX11 drug formulation and Perjeta; fig. 5 (H): trend plots were linearly fitted to the HLX11 drug formulation and CEX acid peak content variation of Perjeta.
Detailed Description
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to be all but practical.
In the experimental examples, the chemical reagents used in the examples are all analytically pure reagents, usually under conventional conditions or under conditions recommended by the manufacturer, and the anti-Her 2 monoclonal antibody may be monoclonal antibody prepared according to any currently known method, and the following exemplary antibody preparation method is provided by Shanghai Fuhonghan biotechnology Co., ltd, and the exemplary method is not limited to the present invention, and the name of the monoclonal antibody is: HLX11 protein. The virus-removed filtered anti-Her 2 monoclonal antibody sample (HLX 11 protein) is concentrated and changed by ultrafiltration, and the replacement liquid to be added is calculated according to the formula of the buffer solution, so that the dilution is completed, the density is defaulted to 1.0g/mL, and the name of the preparation is: HLX11 formulation. The methods of preparing HLX11 formulation products of the present invention are conventional in the art and the exemplary methods are not limiting of the invention.
Filtering the diluted liquid medicine by a 0.22 mu m sterilizing filter, aseptically packaging into 20mL penicillin bottles, adding a 20mm chlorinated butyl rubber plug, and rolling a 20mm aluminum-plastic combined cover. The formula preparation product is used for the following long-term stability test, acceleration stability test and single factor influence test to examine the stability of the finished product, and the storage and transportation conditions are determined.
Example 1 preparation of HLX11 protein
The engineering cell line is constructed by transfecting CHO cells with recombinant plasmid encoding recombinant anti-Her 2 structural domain II humanized monoclonal antibody gene. The production cell culture process comprises seed cell resuscitating, shake flask amplifying, WAVE bioreactor amplifying and one-time reactor production cell culturing, wherein the production cell culturing is carried out by adopting a basal medium containing HM004 and a feed medium containing HF 001. During the culture process, glutamine, a feed medium, glucose and sodium butyrate are supplemented. Culture conditions: viable cell density after inoculation was 0.7X10 6±0.1×106 cells/mL; stirring at 90rpm; pH 6.90+ -0.10; dissolved oxygen is saturated concentration initially, and then is stabilized near the set 40%; the initial temperature is 37.0 ℃; adding sodium butyrate for 6-8 hours, and cooling to 33.0 ℃. Harvested after 15 days of culture or when the cell activity is below 60.0%.
The protein production and purification process flow is characterized in that the protein product is finally obtained through deep filtration, affinity chromatography, low pH virus inactivation, anion chromatography, cation chromatography, virus removal filtration, ultrafiltration concentration and liquid exchange. The protein product is determined to be an anti-Her 2 monoclonal antibody, and the light chain amino acid sequence of the protein product is shown as SEQ ID NO:1, the heavy chain amino acid sequence of which is shown as SEQ ID NO in the sequence table: 2, the resulting protein product was designated HLX11 monoclonal antibody, or HLX11 protein. The preparation of HLX11 proteins can also be found in Stancovski et al PNAS (USA) 88:8691-8695 (1991)), fendly et al cancer Research 50:1550-1558 (1990).
Example 2 screening of buffer System
2.1 First round screening of buffer System
The HLX11 protein obtained in example 1 was prepared as a formulation in Table 1, placed in a thermostatted shaker, shaken at 40℃and 200rpm for 2 weeks, sampled at weeks 0,1 and 2, and assayed for various purposes including protein content (A 280), molecular isomers (SEC-HPLC) and charge isomers (CEX-HPLC). The conditions for high throughput screening studies of HLX11 pharmaceutical formulations are detailed in table 2.
The type of potential buffer system and the pH value range which are helpful for protein stabilization are primarily screened out by a high-throughput screening method. Six common buffer types of acetic acid-sodium acetate, citric acid-sodium citrate, histidine-histidine hydrochloride, histidine-acetic acid, sodium dihydrogen phosphate-disodium hydrogen phosphate and citric acid-disodium hydrogen phosphate are selected to prepare 23 preparation buffers (hereinafter respectively referred to as B1-B23). Wherein, B1-B3 are 20mmol/L acetic acid-sodium acetate buffer systems, and the pH values are 5.5, 6.0 and 6.5 respectively; B4-B7 are 20mmol/L citric acid-sodium citrate buffer system, and the pH values are 5.0, 5.5, 6.0 and 6.5 respectively; B8-B12 are 20mmol/L histidine-histidine hydrochloride buffer systems, B13-B17 are 20mmol/L histidine-acetic acid buffer systems, and the pH values of the two buffer systems are 5.0, 5.5, 6.0, 6.5 and 7.0; the buffer system of sodium dihydrogen phosphate-disodium hydrogen phosphate is 20mmol/L B18-B20, the buffer system of citric acid-disodium hydrogen phosphate is 20mmol/L B21-B23, and the pH values of the two buffer systems are 5.5, 6.0 and 6.5. Of the 23 prescriptions, the histidine-acetic acid buffer systems of B13 to B17 are control prescriptions. The drug concentration for the first study was set at 2.0mg/mL.
Table 1.Hlx11 formulation high throughput buffer system screening study alternative buffer information
TABLE 2 screening study conditions for high throughput buffer systems for HLX11 formulations
The results of the HLX11 high-throughput buffer system screening study are shown in Table 3.
As can be seen from table 3, each test result showed good protein quality at 0 week, with no significant differences between the alternative prescriptions. After shaking for 2 weeks at40 ℃ and 200rpm, the main peak content of each formula SEC showed a decreasing trend compared with 2 weeks, and the decrease of the main peak content was mainly represented by a significant increase of the fragment content. HLX11 pharmaceutical formulation high throughput buffer system screening comparison results for 2 week purity change are shown in figure 1. Wherein, fig. 1 (a): SEC main peak content variation versus plots for different prescriptions; fig. 1 (B): CEX main peak content variation versus plots for different formulations.
According to fig. 1 (a), the decrease in the main peak content of antibody SEC at the same pH in the acetic acid-sodium acetate buffer system, histidine-histidine hydrochloride buffer system, histidine-acetic acid buffer system, and citric acid-disodium hydrogen phosphate buffer system is less than in the citric acid-sodium citrate and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer systems; the main peak change of the antibody SEC under the conditions of each pH value of an acetic acid-sodium acetate buffer system is not different, the main peak content of the antibody SEC in the buffer solution at the pH value of 6.0 in a histidine-histidine hydrochloride buffer system, a citric acid-disodium hydrogen phosphate buffer system and a citric acid-sodium citrate buffer system is slightly smaller than the main peak change of the antibody SEC under the conditions of other pH values of the same group of buffer solutions, and the content change is correspondingly reduced or increased along with the reduction or increase of the pH value; under the conditions of various pH values of a histidine-histidine hydrochloride buffer system, the pH value range which is favorable for stabilizing the molecular isomer can be seen from the molecular isomer result to be 5.5-6.5.
According to the CEX data in Table 3, after shaking at 40℃and 200rpm for 2 weeks, the main peak content of each prescription showed a decrease in the main peak content, which was mainly represented by a significant increase in the acid peak content, compared with 0 weeks. The main peak content of the antibody in the acetic acid-sodium acetate buffer system (A55, A60 and A65), the histidine-histidine hydrochloride buffer system (H55, H60 and H65) and the histidine-acetic acid buffer system (HA 55, HA60 and HA 65) is reduced less under the same pH value condition, and the main peak content of the antibody in the buffer solution at the pH value of 6.0 is slightly less than the change under the other pH value conditions of the same group of buffer solutions. Based on the results of the purity comparison shown in fig. 1 (B) and the data of the change in the purity of the charge isomer, it was determined that the optimum pH range for facilitating the stabilization of the charge isomer was 5.5 to 6.5.
In summary, the high throughput screening study shows that the buffer system contributing to the stability of HLX11 protein is an acetate-sodium acetate buffer system and a histidine-histidine hydrochloride buffer system, and the stability is best when the pH is 5.5 to 6.5 and the pH is 6.0.
Although the high throughput screening shows that the acetic acid-sodium acetate buffer system and the histidine-histidine hydrochloride buffer system are slightly better, the acetic acid system is easy to volatilize, the pKa is 4.8, and the buffer capacity is weaker in the pH range of 5.5-6.5, so the pH range is adjusted to 5.0-6.0 in the next round of research; in addition, the effect of a citric acid-sodium citrate buffer system is further examined; the pH value screening range is fixed to 5.5-6.5 by the common buffer system of histidine-histidine hydrochloride. As a control buffer system, the histidine-acetate buffer system was used to examine only the pH (HA 60) with the best stability.
2.2 Buffer System secondary round screening
The first round of preparation research is to preliminarily screen out candidate buffer types and pH value ranges which are helpful for protein stabilization through a high-throughput screening method. Based on the above, the HLX11 buffer system is further determined by a complete physicochemical analysis means through a high-temperature acceleration test in secondary rounds of research.
Based on the results of the first round of study, 12 buffers (hereinafter referred to as HA60, HA60N, H, H60, H65, a50, a55, a60, C50, C55, C60, and C65, respectively) were selected in total, and HLX11 formulation buffer system screening study candidate buffer information is shown in table 4.
TABLE 4 screening study alternative buffer compositions for HLX11 formulation buffer systems
The protein product of example 1 was taken and prepared as a recipe in Table 4. Filtering the sample in a biosafety cabinet by adopting a disposable sterile filter with the diameter of 0.22 mu m, then aseptically packaging the sample into 2mL penicillin bottles, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. The split samples were placed in a 40 ℃ constant temperature and humidity cabinet for 4 weeks, and were sampled at weeks 0, 1,2 and 4, respectively, and the detection items for detection and analysis included appearance, protein content (a 280), pH, molecular isomer (SEC-HPLC), charge isomer (CEX-HPLC), protein particle size, pdI (DLS) and the like. Screening and researching conditions of the HLX11 preparation buffer system are shown in table 5.
TABLE 5 screening study conditions for HLX11 formulation buffer System
The results of the screening study of the HLX11 formulation buffer system are shown in Table 6 and Table 7.
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As can be seen from table 6, the addition of NaCl resulted in an aggravation of opalescence of the protein solution, and opalescence of each of the prescription solutions under the citric acid-sodium citrate buffer system (C50 to C65) was also slightly more pronounced than the solutions under the other three buffer systems.
As can be seen from the SEC data in Table 7, after acceleration for 4 weeks at 40℃the main peak content of each of the prescriptions SEC tended to decrease compared to 0 weeks, and the decrease in the main peak content was mainly manifested as a significant increase in the fragment content. The main peak content of the antibodies by using the HA60, the HA60N, H, the H60, the A55 and the A60 buffer solutions is reduced consistently; the main peak levels of antibodies in the most acidic C50 buffers decreased slightly faster with the A50, C55, C60 and C65 buffers. Comparison results of 4 week purity change screening of HLX11 pharmaceutical formulation buffer system are shown in fig. 2. Wherein, fig. 2 (a): SEC main peak content variation versus plots for different prescriptions; fig. 2 (B): SEC fragment content variation versus plots for different prescriptions; fig. 2 (C): CEX main peak content variation contrast plots for different prescriptions; fig. 2 (D): CEX acid peak content variation plots for different formulations; fig. 2 (E): comparison of mean particle size variation of DLS proteins for different formulations.
According to the comparison of purity figures 2 (a) - (B) and the data of purity change, histidine-histidine hydrochloride buffer system (pH range 5.5-6.0) is more conducive to molecular isomer stabilization. According to the CEX data of table 7, after acceleration at 40 ℃ for 4 weeks, the main peak content of each prescription showed a decreasing trend compared to 0 weeks, and the decrease of the main peak content was mainly represented by a significant increase of the acid peak content. The main peak of the antibody in the H60 buffer solution is reduced slightly, and the main peaks of the antibody in the HA60, HA60N, H and A60 buffer solutions are reduced slightly, and the main peak of the antibody in the citric acid-sodium citrate buffer system (C50-C65) is reduced faster than the main peak of the antibody in other three buffer systems under the same pH value condition. According to the purity comparison of fig. 2 (C) - (D) and the data on the change in purity of the charge isomer, the histidine-histidine hydrochloride buffer system (pH range 5.5-6.5) is more conducive to the stabilization of the charge isomer.
As can be seen from table 7 and fig. 2 (E), the protein particle size in the histidine-histidine hydrochloride buffer system decreased with increasing pH, but the protein particle sizes in both the acetic acid-sodium acetate buffer system and the citric acid-sodium citrate buffer system were larger than the protein particle size in the histidine-histidine hydrochloride buffer system, and the protein particle sizes in the latter two buffer systems increased with increasing pH, indicating that the higher pH was more likely to produce aggregates. The addition of NaCl to the buffer also resulted in a significant increase in protein particle size.
In conclusion, according to the pH value screening research acceleration stability test result of the buffer system, the buffer effect of stabilizing the pH value of the solution can be achieved under the 20mmol/L histidine-histidine hydrochloride buffer system, and when the pH value is 6.0, the protein stability is the best. We therefore selected a 20mmol/L histidine-HCl buffer system with a pH of 6.0 for subsequent studies.
EXAMPLE 3 HLX11 formulation adjuvant screening study and results
3.1 First round screening of adjuvants
Based on the results of the buffer system screening study, we selected a histidine-histidine hydrochloride system, pH 6.0, as an alternative buffer system for HLX11 pharmaceutical formulations. The study was completed with 6 alternative prescriptions (hereinafter referred to as F1-F6 respectively), F1 was a control prescription, the buffer system was 20mmol/L histidine-acetic acid, and the pH was 6.0, containing 4.1% sucrose and 0.02% polysorbate 20. The buffer system mainly examines a histidine-histidine hydrochloride buffer system, and in addition, pH values (A60 and CP 60) with best stability of two buffer systems, namely acetic acid-sodium acetate and citric acid-sodium citrate, are used as a control buffer system. Stabilizers include sucrose and sorbitol; the surfactant of this round was tentatively 0.02% polysorbate 20.
The round of research designs and screens auxiliary material systems through single-factor experiments, and examines the influence of different buffer liquid types, sodium chloride and stabilizer types on protein stability. The information of the alternative prescriptions for the first round screening study of the HLX11 auxiliary materials is shown in Table 8.
Table 8 hlx11 formulation adjuvant composition for first-round screening study alternative prescriptions
The preparation method comprises the following steps: the HLX11 protein obtained in the example is adopted, three steps of chromatography purification are carried out, auxiliary materials are added after ultrafiltration liquid exchange, and the protein concentration is regulated to prepare the prescription of Table 8. Filtering the sample in a biosafety cabinet by using a disposable sterile filter with the diameter of 0.22 mu m, aseptically packaging 1.0mL of the sample into 2mL of an asepsis penicillin bottle, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. The above split samples were placed in a constant temperature and humidity box at 40deg.C (75% RH) for 4 weeks, and sampled at weeks 0, 2 and 4, respectively, for detection analysis, wherein the detection items include appearance, protein content (A 280), pH, molecular isomer (SEC-HPLC), protein particle size and PdI (DLS), charge isomer (CEX-HPLC), etc. The screening and researching conditions of the HLX11 preparation auxiliary materials are shown in Table 9.
TABLE 9 first round screening study investigation conditions for HLX11 adjuvant
The results of the first round screening study of HLX11 auxiliary materials are shown in Table 10.
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As can be seen from table 10, the addition of NaCl in recipe HSuN resulted in a heavier opalescence of the protein solution, and the opalescence of the recipe C60 protein solution was also slightly more pronounced than the solutions under the other two sets of buffer systems. The comparison results of 4-week purity change of HLX11 pharmaceutical formulation adjuvant screening are shown in fig. 1. Wherein, fig. 3 (a): SEC main peak content change trend comparison graphs of different prescriptions; fig. 3 (B): a SEC fragment content change trend comparison chart of different prescriptions; fig. 3 (C): CEX main peak content change trend comparison diagrams of different prescriptions; fig. 3 (D): CEX acidic peak content change trend comparison graphs of different prescriptions; fig. 3 (E): comparison of mean particle size variation of DLS proteins for different formulations.
As can be seen from the combination of table 10 and the SEC data in fig. 3 (a) to (B), after acceleration at 40 ℃ for 4 weeks, the main peak content of each prescription SEC showed a decreasing trend compared with 0 weeks, and the decrease of the main peak content was mainly represented by a significant increase of the fragment content. The main peak levels of antibodies in formulas HASu and HSuN decreased faster than the other formulas, while the main peak levels of antibodies in formulas HSu, HSo, ASu and CSu decreased nearly; prescription HSo antibody main peak content decreased slightly slower than HSu.
In combination with the CEX data of table 10 and fig. 3 (C) - (D), after acceleration at 40 ℃ for 4 weeks, the main peak content of each prescription showed a decreasing trend compared to 0 weeks, and the decrease of the main peak content was mainly represented by a significant increase of the acid peak content. The main peak levels of antibodies in formulas HASu and CSu decreased faster than the other formulas, the main peak level of antibodies in formula HSuN decreased slightly slower, while the main peak levels of antibodies in formulas HSu, HSo and ASu decreased consistently and slowest.
As can be seen from Table 10 and FIG. 3 (E), the addition of NaCl to the formulation HSuN also resulted in a significant increase in protein particle size; the larger particle size of the protein in the ASu and CSu formulations compared to histidine-histidine hydrochloride buffer system further demonstrates that the acetate-sodium acetate buffer system and the citrate-sodium citrate buffer system are prone to generate aggregates.
In conclusion, according to the results of the accelerated stability test of the first round of auxiliary material screening, when the pH value of the 20mmol/L histidine-histidine hydrochloric acid buffer system is 6.0, 3.0% sorbitol is added as a stabilizer, and the protein stability is best. Therefore, we selected a histidine-hcl buffer system with 20mmol/L pH of 6.0 and 3.0% sorbitol as stabilizer for subsequent surfactant type and content studies.
3.2 Auxiliary Material secondary screening
The round of research further designs and screens auxiliary material systems through single-factor experiments, and examines the influences of different surfactant types and contents. HLX11 adjuvant screening alternative prescription information for the next round of study is shown in table 11.
Table 11 hlx11 formulation adjuvant alternative formulation composition for round-robin screening studies
The preparation method comprises the following steps: the HLX11 protein obtained in example 1 was purified by three steps of chromatography, ultrafiltration and protein concentration was adjusted by adding adjuvants to the obtained solution. Filtering the sample in a biosafety cabinet by using a disposable sterile filter with the diameter of 0.22 mu m, aseptically packaging 1.0mL of the sample into 2mL of an asepsis penicillin bottle, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. The above-mentioned split samples were individually shaken in a constant temperature shaker at 40℃and 75% RH for 4 weeks at 200rpm, and sampled at 0 th, 1 th, 2 nd and 4 th weeks, respectively, and the detection items include appearance, protein content (A 280), pH, molecular isomer (SEC-HPLC), charge isomer (CEX-HPLC), protein particle size, and PdI (DLS) and the like. HLX11 formulation adjuvant screening the conditions for the rounds of study and investigation are detailed in table 12.
Table 12.HLX11 adjuvant secondary screening study conditions
The results of the HLX11 adjuvant secondary screening study are shown in Table 13.
TABLE 13 data summary of HLX11 formulation adjuvant run-around screening study accelerated stability test (200 rpm at 40 ℃ C.)
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As shown in Table 13, each test result showed good protein quality at 0 weeks, with no significant differences for each alternative prescription. After 4 weeks of examination at 40℃and 200rpm acceleration, the basic physicochemical properties (appearance, protein content and pH) of each formulation were not significantly changed from the average particle size of the protein and PdI.
The SEC data showed that the main peak levels of each of the prescribed antibodies SEC tended to decrease with no significant change in the polymer, with the main peak levels decreasing primarily as a significant increase in the fraction levels, with the main peaks of the prescribed antibodies F1 (without polysorbate 20 or 80) and F2 (with 0.02% polysorbate 20) decreasing minimally, while the main peaks of the antibodies SEC for F3 (with 0.05% polysorbate 20) and F4 (with 0.02% polysorbate 80) decreasing slightly faster.
CEX data showed that the main peak content of CEX antibody was decreasing with no significant change in alkali peak, and the decrease in CEX main peak content was mainly manifested as a significant increase in acid peak content, with the main peak decrease for the prescription F1 (without polysorbate 20 or 80) antibody being minimal, the main peak decrease for the F2 (with polysorbate 20 at 0.02%) antibody being slightly faster, and the main peak decrease for the F3 (with polysorbate 20 at 0.05%) and F4 (with polysorbate 80 at 0.02%) antibody being the fastest. Comparison results of 4-week purity change of HLX11 formulation surfactant screening are shown in fig. 4. Wherein, FIG. 4 (A) is a graph showing the variation of SEC main peak content; FIG. 4 (B) is a graph showing the variation of SEC fragment content; FIG. 4 (C) is a graph showing the comparison of the CEX main peak content; FIG. 4 (D) is a graph showing the comparison of CEX acidic peak content.
In summary, protein stability was slightly better when 0.02% ps20 was added than when 0.02% ps80 was added to the formulation, in combination with SEC purity and CEX purity variation vs. (fig. 4 (a) - (D)). When PS20 concentration was 0.00%, 0.02% and 0.05% was added to the recipe (recipes F1 to F3), there was no obvious difference in protein stability, and all alternative recipes were good in stability. Therefore, the content of PS20 in the HLX11 formulation prescription can be controlled within the range of 0.20+/-0.2 mg/mL.
Through formulation prescription development, we screened out buffer systems and auxiliary materials which help to stabilize the HLX11 protein, and determined that the prescription composition of the HLX11 pharmaceutical formulation is: 30mg/mLHLX11 of monoclonal antibody, 1.51mg/mL of histidine, 2.15mg/mL of histidine hydrochloride (molar concentration of histidine-histidine hydrochloride buffer: 20 mmol), 30.0mg/mL of sorbitol, 0.2mg/mL of polysorbate 20 (volume percentage content of polysorbate 20: 0.02%), and pH value of 6.0.
EXAMPLE 4 HLX11 formulation and Perjeta preliminary stability control study
HLX11 protein replacement fluid obtained using the final culture and purification process was diluted to the final prescription as described in example 1, HLX11 drug formulation and Perjeta (purchased from roche company) were each filtered in a biosafety cabinet with a 0.22 μm disposable sterile filter, aseptically dispensed 1.0mL of sample into 2mL sterile penicillin bottles, 13mm plug was added, and 13mm aluminum plastic composite cap was rolled using the same inner packaging material and specifications.
The formula of the Perjeta raw grinding preparation is as follows: 30mg/mL Pertuzumab, 20mM L-histidine acetate, 120mM sucrose and 0.02% polysorbate 20 at pH 6.0.
The investigation conditions are 50 ℃, the sample is placed for 1 month, and the samples are respectively sampled and detected at 0 week, 1 week, 2 weeks and 4 weeks, wherein the detection items comprise appearance, protein content, osmolality, purity (SEC-HPLC, CEX-HPLC, CE-SDS) and the like. HLX11 pharmaceutical formulations and Perjeta preliminary stability control the conditions are investigated in table 14.
TABLE 14 HLX11 pharmaceutical formulations and Perjeta preliminary stability control experiments conditions were investigated
The results of HLX11 vs. Perjeta preliminary stability controls are detailed in Table 15.
TABLE 15 HLX11 pharmaceutical formulations and Perjeta preliminary stability control study results
Results of HLX11 drug formulation preliminary stability study acceleration test SEC and CEX individual component content variation are shown in fig. 5. Wherein, FIG. 5 (A) is a SEC main peak content variation trend chart; FIG. 5 (B) is a graph showing the variation trend of the SEC fragment content; FIG. 5 (C) is a plot of the linear fit trend of SEC main peak content variation; FIG. 5 (D) is a plot of the linear fit trend of the SEC fragment content variation; FIG. 5E shows the variation trend of the CEX main peak content; FIG. 5F shows the variation trend of CEX acidic peak content; FIG. 5 (G) is a plot of the linear fit trend of CEX main peak content variation; FIG. 5 (H) shows a linear fit trend of CEX acid peak content variation.
The main peak content of each of the antibodies SEC of the prescriptions showed a decreasing trend in combination with the data of Table 15 and FIGS. 5 (A) to (D), and the decrease of the main peak content was mainly represented by a significant increase of the fragment content. The linear fitting equations of the SEC main peak content change of HLX11 and Perjeta are y= -2.25x+102.2 and y= -2.20x+102.2 respectively, and the difference between the main peak falling slope-2.25 of HLX11 and the main peak falling slope-2.2 of Perjeta is small, so that the SEC main peak falling trend of HLX11 and Perjeta is consistent. The linear fit equations for the SEC fragment content variation of HLX11 and Perjeta are y=2.05x+2.4 and y=1.85x+1.95, respectively, and the SEC fragment increase slope of HLX11 is 2.05 greater than the fragment increase slope of Perjeta by 1.85, so that the SEC fragment increase trend of HLX11 is slightly faster than the fragment increase trend of Perjeta. However, after 4 weeks of examination at 50℃the SEC fragments increased by 6.4% (HLX 11) and 5.5% (Perjeta), respectively, with negligible differences.
Table 15 and CEX data of FIGS. 5 (E) - (H) show that the main peak content of CEX antibody for each prescription is in a decreasing trend, the alkali peak is not changed obviously, and the decrease of CEX main peak content is mainly shown as the significant increase of acid peak content. The linear fitting equations of the HLX11 and the Perjeta CEX main peak content change are y= -16.39x+92.9 and y= -17.27x+90.55 respectively, and the main peak falling slope-16.39 of the HLX11 is slightly larger than the main peak falling slope-17.27 of the Perjeta, so that the main peak falling trend of the HLX11CEX is slightly slower than that of the Perjeta. The linear fit equations for the CEX acid peak content variation of HLX11 and Perjeta are y=15.89x+3.55 and y=18.59 x-4.95, respectively, and the acid peak increase slope 15.89 of HLX11 is significantly smaller than the acid peak increase slope 18.59 of Perjeta, so that the acid peak increase trend of HLX11 is significantly slower than that of Perjeta.
In conclusion, the HLX11 pharmaceutical formulation obtained by the invention has slightly lighter opalescence than the Perjeta formulation, and the osmotic pressure is more similar to that of human blood; the SEC purity change trend of the HLX11 preparation is consistent with that of the Perjeta preparation; the main peak decrease trend of HLX11CEX was slightly slower than that of Perjeta, and the peak increase trend of HLX11CEX acid was significantly weaker than that of Perjeta acid. Compared with the original ground drug Perjeta preparation, the HLX11 drug preparation is more beneficial to antibody stabilization, and can ensure the validity period of more than four weeks under the storage condition of not higher than 50 ℃.
It will be appreciated that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the application, and that such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Sequence listing
<110> Shanghai complex Han dynasty, inc
Shanghai Fu Honghan Lin biopharmaceutical Co., ltd
<120> A pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody
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Claims (4)

1. A pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody, the pharmaceutical formulation comprising: anti-Her 2 monoclonal antibodies, buffers, protein protectants, and surfactants;
wherein the light chain amino acid sequence of the anti-Her 2 monoclonal antibody is shown as SEQ ID NO. 1 in a sequence table, the heavy chain amino acid sequence of the anti-Her 2 monoclonal antibody is shown as SEQ ID NO. 2 in the sequence table, and the protein concentration of the anti-Her 2 monoclonal antibody is 5mg/ml-50mg/ml;
wherein the buffer solution is histidine-histidine hydrochloride buffer solution, and the concentration of the buffer solution is 20 mM;
Wherein the protein protecting agent is sorbitol, and the mass percentage content of the protein protecting agent is 3%; and is also provided with
Wherein the surfactant is polysorbate 20, and the content of the surfactant is 0.1 mg/mL-0.4 mg/mL;
Wherein the pharmaceutical formulation has a pH of 5.5-6.5.
2. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises: 30mg/mL of anti-Her 2 monoclonal antibody, 20 mmol/L of histidine-histidine hydrochloride buffer, 3% sorbitol by mass, 0.2mg/mL of polysorbate 20, pH 6.0.
3. The pharmaceutical formulation according to claim 1 or 2, wherein the pharmaceutical formulation is a liquid formulation for injection.
4. A lyophilized formulation of the pharmaceutical formulation of claim 1 or 2.
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CN112798720B (en) * 2021-01-11 2022-04-19 苏州药明生物技术有限公司 Application of histidine buffer solution in reduction of protein aggregates
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102319430A (en) * 2004-10-20 2012-01-18 健泰科生物技术公司 Antibody formulations in histidine-acetate buffer
CN102416176A (en) * 1995-07-27 2012-04-18 基因技术股份有限公司 Stabile isotonic lyophilized protein formulation

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CN101199483B (en) * 2006-12-14 2011-01-26 上海中信国健药业股份有限公司 Stable anti-HER2 humanized antibody preparation
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CN102309754A (en) * 2010-07-07 2012-01-11 上海中信国健药业股份有限公司 Stable medicinal composition of recombinant humanized antibody

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CN102416176A (en) * 1995-07-27 2012-04-18 基因技术股份有限公司 Stabile isotonic lyophilized protein formulation
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