CN111375057A

CN111375057A - Pharmaceutical formulation comprising anti-Her 2 monoclonal antibody

Info

Publication number: CN111375057A
Application number: CN201811628635.0A
Authority: CN
Inventors: 马崇军; 韩冬梅; 姜伟东
Original assignee: Shanghai Fuhong Hanlin Biopharmacy Co ltd; Shanghai Henlius Biotech Inc
Current assignee: Shanghai Fuhong Hanlin Biopharmacy Co ltd; Shanghai Henlius Biotech Inc
Priority date: 2018-12-28
Filing date: 2018-12-28
Publication date: 2020-07-07

Abstract

The present invention provides a pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody, said pharmaceutical formulation comprising: an anti-Her 2 monoclonal antibody, a buffer solution, an isoosmotic adjusting agent, a protein protective agent, a surfactant and the like. The product is stored for a certain time at room temperature 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 preparation obtained by the invention can ensure the validity period of more than four weeks under the condition of not higher than 50 ℃. Compared with the original medicine preparation, the medicine preparation has better stability and is more beneficial to antibody stabilization.

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 (Pertuzumab, trade name: Perjeta) is a recombinant humanized monoclonal antibody targeting HER 2. The drug is first approved by the U.S. FDA to be marketed in 2012 for use in combination with trastuzumab and docetaxel to treat Her2 positive metastatic breast cancer patients who have no history of prior anti-Her 2 treatment or chemotherapy treatment of metastatic disease.

Pharmaceutical preparations based on proteins (monoclonal antibodies and the like) generally comprise active ingredients, buffer ingredients, isotonicity adjusting agents, stabilizers and the like. The saccharides or alcohols are common medicine adding auxiliary materials, mainly play a role of a stabilizer, have the characteristics of good stability, no accumulation in human bodies, safety and the like, can help biomacromolecule medicines to maintain the molecular structures and the biological activities of the biomacromolecule medicines, and are favorable for the long-term stability of the medicines. Sodium chloride is used as an isoosmotic adjusting agent, and a small amount of sodium chloride is added into a protein solution, so that the charge on the surface of a protein molecule is increased, the action of the protein molecule and a water molecule is enhanced, and the solubility of the protein in an 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 the aggregation of protein on a two-phase interface 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 medicines comprise basic physicochemical property detection, protein purity detection, particle detection, protein activity detection and the like. The basic physical and chemical property detection items comprise appearance, pH value, protein concentration, osmotic pressure molar concentration and the like, the protein purity detection comprises detection of molecular isomers (SEC-HPLC, SDS-PAGE) and charge isomers (CEX-HPLC, cIEF) and the like, and the particle detection generally adopts imaging and counting of sub-visible particles, Flowcam, dynamic light scattering DLS, a light resistance method and the like.

Pharmaceutical preparations of the protein (monoclonal antibody, etc.) type are suitable for parenteral administration, including intravenous, intramuscular, intraperitoneal or subcutaneous injection. The protein in the liquid preparation is easy to form polymer or particles, which is always a problem of the preparation of protein drugs. In general, it is difficult to maintain good physical, chemical and biological stability of proteins, especially monoclonal antibody drugs, contained in pharmaceutical preparations during storage.

Disclosure of Invention

In order to provide protein formulations with good stability that meet the needs of the pharmaceutical industry, the present invention provides a novel pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody. And a protein preparation with higher stability is obtained through high-throughput screening and formula optimization research.

The buffer system was screened to examine its effect on protein stability. And designing a single-factor test to investigate the influence of different ionic strengths, protein concentrations, pH values, stabilizer types and surfactant types on the stability of the protein.

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, said pharmaceutical formulation comprising: an anti-Her 2 monoclonal antibody, a buffer, a protein protectant, and a surfactant.

Wherein said "pharmaceutical formulation" is conventional in the art and generally refers to a preparation in such a form that allows the biological activity of the active ingredient to be effective, the subject to be well adapted, to meet clinical needs and not to 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 may be reasonably administered to a subject mammal to provide an effective dose.

The dosage form of the pharmaceutical formulation is a conventional dosage form in the art, and preferably includes a liquid preparation for injection or a lyophilized preparation, and the like. 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, etc., and 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 art, more preferably a recombinant anti-Her 2 fully human monoclonal antibody, the anti-Her 2 monoclonal antibody is preferably Pertuzumab (Pertuzumab), and preferably the HLX11 monoclonal antibody prepared by the invention. The light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 1 in a sequence table, and the heavy chain amino acid sequence thereof 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 30 mg/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: the engineering cell line is constructed by transfecting CHO cells with recombinant plasmids of genes encoding recombinant anti-Her 2 domain II humanized monoclonal antibodies.

The production cell culture process includes seed cell recovery, shake flask amplification, WAVE bioreactor amplification and disposable reactor production cell culture, and adopts basic culture medium containing HM004 and supplementary culture medium containing HF001 to culture production cells, and during the culture, glutamine, supplementary culture medium, glucose and sodium butyrate are added, and the culture condition is that the density of inoculated living cells is 0.7 × 10⁶±0.1×10⁶cells/mL; the stirring speed is 90 rpm; pH 6.90 +/-0.10; the dissolved oxygen is at the saturation concentration initially and then is stabilized to be about 40 percent of the set value; initial temperature 37.0 ℃; adding sodium butyrate for 6-8 h, and cooling to 33.0 ℃. And (4) harvesting after 15 days of culture or when the activity of the cells is lower than 60.0%.

The protein production and purification process comprises deep filtration, affinity chromatography, low pH virus inactivation, anion chromatography, cation chromatography, virus removal filtration, ultrafiltration concentration and liquid exchange, preparing the final protein product into the formula shown in Table 1, placing in a constant temperature shaking table, shaking at 40 deg.C and 200rpm for 2 weeks, sampling at 0, 1 and 2 weeks, detecting and analyzing, wherein the detection items comprise protein content (A)₂₈₀) Molecular isomers (SEC-HPLC) and charge isomers (CEX-HPLC).

Where the buffer is one conventional in the art, "buffer" generally refers to a buffered solution that resists changes in pH by the action of its acid-base complexing components. Preferably, the buffer solution of the present invention comprises: one of buffer solution systems such as citric acid-sodium citrate, histidine-histidine hydrochloride, acetic acid-sodium acetate or citric acid-disodium hydrogen phosphate; more preferably citric acid-sodium citrate or histidine-histidine hydrochloride buffer solution system; most preferred is a histidine-histidine hydrochloride buffer system. The concentration of the buffer is preferably: 10-30mM, more preferably 15-25mM, preferably 20 mM.

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 like, and the preparation method of the anti-Her 2 monoclonal antibody is the technical content known by the technical personnel in the field.

Wherein the protein protectant is a protein protectant as is conventional in the art, or referred to as a "protein stabilizer". The protein protectant can protect the stability of the protein drug and protect the function of the protein drug from changes in conditions (e.g., freezing, lyophilization, or other manufacturing conditions). The protein protectant preferably includes a saccharide, protein, amino acid, polymer, salt, amine, surfactant, etc., more preferably includes one or more of sorbitol, sucrose, trehalose, mannitol, arginine hydrochloride or glycine; sorbitol is preferred. The protein protective agent preferably comprises the following components in percentage by mass: 1% to 5%, more preferably 2% to 4%, preferably 3%.

Wherein the surfactant is a surfactant conventional in the art, preferably a nonionic surfactant. Examples of the surfactant 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-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearoyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristoamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g. lauramidopropyl betaine); myristamidopropyl-, palmitoylaminopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; the MONAQUATTM series (Mona industries Inc, Paterson, NJ); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68), 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 to 0.4mg/mL, more preferably 0.15mg/mL to 0.35mg/mL, and preferably 0.2 mg/mL.

The pH of the pharmaceutical formulation of the present invention is a pH conventional in the art, preferably: a pH of 5-7, more preferably: a pH of 5.5-6.5, preferably: pH 6.0.

The pharmaceutical formulation of the present invention may or may not contain an isotonic regulator as required, wherein the isotonic regulator is a conventional isotonic regulator in the art, preferably glucose or sodium chloride, and 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 solution, 3% by mass of sorbitol, 0.2mg/mL of polysorbate 20, and the pH value of the solution is 6.0.

The pharmaceutical formulations of the present invention are in the form of dosage forms conventional in the art. Preferably including liquid preparations, solid preparations, lyophilized preparations, etc.; more preferably, the pharmaceutical formulation is a liquid preparation for injection or a lyophilized preparation.

The pharmaceutical preparation prepared by the invention is stored for a certain time at room temperature or higher temperature (40-50 ℃), all main detection indexes have no obvious change, and the pharmaceutical 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 in the pharmaceutical formulation, and can ensure the effective period of more than four weeks under the storage condition of not higher than 50 ℃.

Drawings

FIG. 1: HLX11 pharmaceutical formulations high throughput buffer systems were screened for 2-week purity change comparisons. Wherein, fig. 1 (a): contrast graph of SEC main peak content variation of different prescriptions; FIG. 1 (B): CEX main peak content variation comparative graph of different prescriptions.

FIG. 2: HLX11 pharmaceutical formulation buffer system screening for 4-week purity change comparison results. Wherein, fig. 2 (a): contrast graph of SEC main peak content variation of different prescriptions; fig. 2 (B): graph comparing the content change of SEC fragments of different prescriptions; fig. 2 (C): comparing the content change of the CEX main peak of different prescriptions; fig. 2 (D): CEX acid peak content variation comparative graphs of different prescriptions; fig. 2 (E): graph comparing the change of the average particle size of DLS protein of different prescriptions.

FIG. 3: HLX11 pharmaceutical formulation excipients screening for 4-week purity change comparison results. Wherein, fig. 3 (a): SEC main peak content variation trend comparison graphs of different prescriptions; fig. 3 (B): SEC fragment content trend comparison graph of different prescriptions; fig. 3 (C): comparing the content variation trend of the CEX main peak of different prescriptions; fig. 3 (D): a CEX acidic peak content variation trend comparison graph of different prescriptions; fig. 3 (E): graph comparing the change of the average particle size of DLS protein of different prescriptions.

FIG. 4: HLX11 formulation surfactant screening 4 weeks purity change comparison results. Wherein, fig. 4(a): contrast graph of SEC main peak content variation of different prescriptions; fig. 4(B): graph comparing the content change of SEC fragments of different prescriptions; fig. 4(C): comparing the content change of the CEX main peak of different prescriptions; fig. 4(D): CEX peak content variation of different prescriptions is compared with the graph.

FIG. 5: initial stability studies of HLX11 pharmaceutical formulations accelerated the results of testing SEC and CEX for changes in the content of each component. Fig. 5(a): graphs comparing the trend of the SEC major peak content for HLX11 pharmaceutical formulations and Perjeta; fig. 5(B): a graph of SEC fragment content trend for HLX11 pharmaceutical formulations and Perjeta; fig. 5(C): linearly fitting trend plots for SEC major peak content changes for HLX11 pharmaceutical formulations and Perjeta; fig. 5(D): the SEC fragment content changes for HLX11 pharmaceutical formulations and Perjeta were linearly fitted to a trend graph. Fig. 5(E): a trend plot of the CEX main peak content for the HLX11 pharmaceutical formulation and Perjeta; fig. 5(F): CEX acid peak content trend plots for HLX11 pharmaceutical formulations and Perjeta; fig. 5(G): linearly fitting trend plots for the change in CEX major peak content for the HLX11 pharmaceutical formulation and Perjeta; fig. 5(H): the CEX acid peak content changes for the HLX11 pharmaceutical formulation and for Perjeta were linearly fitted to a trend plot.

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 that the experiments below are all experiments that have been performed and that are only experiments that can be performed.

The experimental method without specifying the specific conditions in the experimental examples, generally according to the conventional conditions or according to the conditions suggested by the manufacturers, the chemical reagents used in the examples are all analytical pure reagents, the anti-Her 2 monoclonal antibody can be a monoclonal antibody prepared according to any method known so far, and the following exemplary antibody preparation method is provided by the company of shinkanglin biotechnology gmbh of shanghai, which is not intended to limit the present invention, and the name of the monoclonal antibody is: HLX11 protein. An anti-Her 2 monoclonal antibody sample (HLX11 protein) after virus removal filtration is subjected to ultrafiltration concentration liquid exchange, according to a buffer solution formula, replacement liquid needing to be supplemented is calculated, dilution is completed, the density is 1.0g/mL by default, and the preparation is named as: HLX11 formulation. The methods of preparation of the HLX11 formulated products described herein are conventional in the art and are not intended to be limiting.

Filtering the diluted liquid medicine by a 0.22 mu m sterilizing filter, aseptically subpackaging into 20mL penicillin bottles, adding 20mm chlorinated butyl rubber plugs, and rolling 20mm aluminum-plastic combined covers. The formulated product is used for long-term stability test, accelerated stability test and single factor influence test below to investigate the stability of the finished product and determine the storage and transportation conditions.

Example 1 preparation of HLX11 protein

The engineering cell line is constructed by transfecting CHO cells with recombinant plasmids of genes encoding recombinant anti-Her 2 structural domain II humanized monoclonal antibodies. The production cell culture process comprises seed cell recovery, shake flask amplification, WAVE bioreactor amplification and disposable reactor production cell culture,adopting a basic culture medium containing HM004 and a supplementary culture medium containing HF001 to culture the production cells, supplementing glutamine, the supplementary culture medium, glucose and sodium butyrate in the culture process, and adopting the culture conditions that the density of the inoculated living cells is 0.7 × 10⁶±0.1×10⁶cells/mL; the stirring speed is 90 rpm; pH 6.90 +/-0.10; the dissolved oxygen is at the saturation concentration initially and then is stabilized to be about 40 percent of the set value; initial temperature 37.0 ℃; adding sodium butyrate for 6-8 h, and cooling to 33.0 ℃. And (4) harvesting after 15 days of culture or when the activity of the cells is lower than 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, and finally the protein product is obtained. Determining that the obtained protein product is an anti-Her 2 monoclonal antibody, wherein the light chain amino acid sequence of the monoclonal antibody is shown as SEQ ID NO:1, and the heavy chain amino acid sequence thereof is shown as SEQ ID NO:2, the obtained protein product is named as HLX11 monoclonal antibody or HLX11 protein. The preparation method of HLX11 protein can also refer to the technical contents disclosed in Stancovski et al, PNAS (USA)88: 8691-.

Example 2 screening of buffer systems

2.1 first round screening of buffer systems

The HLX11 protein obtained in example 1 is prepared into the formula shown in Table 1, placed in a constant temperature shaking table, shaken at 40 ℃ and 200rpm for 2 weeks, sampled at 0, 1 and 2 weeks respectively, detected and analyzed, and the detection items comprise protein content (A)₂₈₀) Molecular isomers (SEC-HPLC) and charge isomers (CEX-HPLC). HLX11 pharmaceutical formulations high throughput screening study examination conditions are detailed in table 2.

The potential buffer system type and the pH value range which are beneficial to protein stabilization are preliminarily screened out by a high-throughput screening method. A total of 23 formulation buffers (hereinafter referred to as B1-B23) were prepared from six types of common buffers, 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. Wherein, B1-B3 are 20mmol/L acetic acid-sodium acetate buffer system, 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; B18-B20 are 20mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer systems, B21-B23 are 20mmol/L citric acid-disodium hydrogen phosphate buffer systems, and the pH values of the two buffer systems are 5.5, 6.0 and 6.5. Among the 23 prescriptions, histidine-acetic acid buffer systems of B13-B17 are used as reference prescriptions. The drug concentration for the first study was set at 2.0 mg/mL.

TABLE 1 HLX11 formulation high throughput buffer System screening study candidate buffer information

TABLE 2 HLX11 formulation high throughput buffer system screening study examination conditions

The results of the HLX11 high-throughput buffer screening study are detailed in Table 3.

As can be seen from Table 3, the results of the tests all showed good protein quality at week 0, and there was no significant difference between the alternative prescriptions. After shaking for 2 weeks at 40 ℃ and 200rpm, the SEC main peak content at each place is in a descending trend compared with 2 weeks, and the reduction of the main peak content is mainly shown as the obvious increase of the fragment content. HLX11 pharmaceutical formulations high throughput buffer system screening 2 weeks purity change comparison results are shown in figure 1. Wherein, fig. 1 (a): contrast graph of SEC main peak content variation of different prescriptions; FIG. 1 (B): CEX main peak content variation comparative graph of different prescriptions.

According to fig. 1(a), compared to the citric acid-sodium citrate and sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system, the decrease of the main peak content of the antibody SEC under the same pH condition in the acetic acid-sodium acetate buffer system, the histidine-histidine hydrochloride buffer system, the histidine-acetic acid buffer system, and the citric acid-disodium hydrogen phosphate buffer system is less; the change of the main antibody SEC peak is not different under each pH value condition of an acetic acid-sodium acetate buffer system, the content of the main antibody SEC peak in the buffer solution with 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 less than that of the buffer solution in the same group under other pH value conditions, and the content change is correspondingly reduced or increased along with the reduction or increase of the pH value; under various pH value conditions in a histidine-histidine hydrochloride buffer system, the molecular isomer result shows that the pH value range which is favorable for the stability of the molecular isomer is 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 in each square showed a decrease trend, and the decrease in the main peak content was mainly manifested as a significant increase in the acid peak content, compared with 0 week. The content of the main peak of the antibody in the buffer solution with the pH value of 6.0 is slightly less than that in the buffer solutions with the pH values of the same group under the same pH value condition in an acetic acid-sodium acetate buffer system (A55, A60 and A65), a histidine-histidine hydrochloride buffer system (H55, H60 and H65) and a histidine-acetic acid buffer system (HA55, HA60 and HA 65). Based on the purity comparison results shown in FIG. 1(B) and the charge isomer purity change data, the optimum pH range contributing to the stabilization of the charge isomer is determined to be 5.5 to 6.5.

In conclusion, high throughput screening studies show that the buffer systems contributing to the stability of the HLX11 protein are an acetic acid-sodium acetate buffer system and a histidine-histidine hydrochloride buffer system, the pH range is 5.5-6.5, and the stability is the best when the pH is 6.0.

Although high-throughput screening shows that an acetic acid-sodium acetate buffer system and a histidine-histidine hydrochloride buffer system are slightly good, the acetic acid system is easy to volatilize, the pKa is 4.8, and the buffer capacity is weak in the pH range of 5.5-6.5, so that 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 inspected; the pH value of the histidine-histidine hydrochloride which is a common buffer system is fixed within the range of 5.5-6.5. Histidine-acetic acid buffer was used as a control buffer and only the pH with the best stability was examined (HA 60).

2.2 buffer System second round screening

The first round of preparation research preliminarily screens candidate buffer types and pH value ranges which are beneficial to protein stabilization through a high-throughput screening method. On the basis, the HLX11 buffer system is further determined by a complete physicochemical analysis means through a high-temperature acceleration test in a secondary research.

According to the results of the first round of study, 12 buffers (hereinafter referred to as HA60, HA60N, H55, H60, H65, a50, a55, a60, C50, C55, C60 and C65) were selected in the round of study, and information of candidate buffers for HLX11 formulation buffer screening study is shown in table 4.

TABLE 4 HLX11 formulation buffer System screening study candidate buffer compositions

The protein product of example 1 was prepared as shown in Table 4. Filtering the sample by adopting a disposable sterile filter with the diameter of 0.22 mu m in a biological safety cabinet, then aseptically subpackaging the sample into 2mL penicillin bottles, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. Placing the subpackaged samples in a constant temperature and humidity box at 40 deg.C for 4 weeks, sampling at 0, 1, 2 and 4 weeks, detecting and analyzing, wherein the detection items include appearance and protein content (A)₂₈₀) pH, molecular isomer (SEC-HPLC), charge isomer (CEX-HPLC), protein particle size and PdI (DLS), etc. HLX11 formulation buffer screening study examination conditions are shown in Table 5.

TABLE 5 HLX11 formulation buffer screening study examination conditions

Results of the HLX11 formulation buffer screening study are detailed in tables 6 and 7.

As can be seen from Table 6, the addition of NaCl resulted in an increased opalescence of the protein solution, and the opalescence of the solutions at various positions under the citric acid-sodium citrate buffer system (C50-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 4 weeks of acceleration at 40 ℃, the SEC main peak content at each side showed a decreasing trend compared with 0 weeks, and the decrease in the main peak content was mainly manifested as a significant increase in fragment content. The main peak content of the antibody is reduced uniformly by using HA60, HA60N, H55, H60, A55 and A60 buffer solutions; the content of the main antibody peak is reduced slightly quickly by using the buffers A50, C55, C60 and C65, and the content of the main antibody peak is reduced most quickly in the most acidic buffer C50. The results of the purity change comparison of HLX11 pharmaceutical formulation buffer screening for 4 weeks are shown in figure 2. Wherein, fig. 2 (a): contrast graph of SEC main peak content variation of different prescriptions; fig. 2 (B): graph comparing the content change of SEC fragments of different prescriptions; fig. 2 (C): comparing the content change of the CEX main peak of different prescriptions; fig. 2 (D): CEX acid peak content variation comparative graphs of different prescriptions; fig. 2 (E): graph comparing the change of the average particle size of DLS protein of different prescriptions.

According to the comparison of the purity figures 2(A) - (B) and the purity change data, a histidine-histidine hydrochloride buffer system (pH value ranging from 5.5 to 6.0) is more helpful for stabilizing the molecular isomer. According to the CEX data in Table 7, after 4 weeks of acceleration at 40 ℃, the main peak content of each square showed a decrease tendency as compared with 0 weeks, and the decrease of the main peak content was mainly manifested as a significant increase in the acid peak content. The main peak of the antibody in the H60 buffer solution is reduced least, the main peak of the antibody in the HA60, HA60N, H55 and A60 buffer solutions is reduced slightly faster, and the main peak of the antibody in the citric acid-sodium citrate buffer system (C50-C65) is reduced faster than that in the other three groups of buffer systems under the same pH value condition. According to the purity comparison data of FIGS. 2(C) to (D) and the charge isomer purity change data, the histidine-histidine hydrochloride buffer system (pH value range of 5.5-6.5) is more helpful for stabilizing the charge isomer.

As is clear 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 size in both the acetic acid-sodium acetate buffer system and the citric acid-sodium citrate buffer system was larger than that in the histidine-histidine hydrochloride buffer system, and the protein particle size in the latter two buffer systems increased with increasing pH, indicating that higher pH is more likely to generate aggregates. The addition of NaCl to the buffer also resulted in a significant increase in protein particle size.

In summary, according to the results of the accelerated stability test of the buffer system, the buffer system can play a role in stabilizing the pH value of the solution under a 20mmol/L histidine-histidine hydrochloric acid buffer system, and when the pH value is 6.0, the protein stability is the best. Therefore, we chose 20mmol/L histidine-histidine hydrochloride buffer system at pH6.0 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 the histidine-histidine hydrochloride system, pH6.0, as an alternative buffer system for HLX11 pharmaceutical formulations. In the present round, 6 candidate formulations (hereinafter referred to as F1-F6, respectively) were prepared, F1 was used as a control formulation, the buffer system was 20mmol/L histidine-acetic acid, and the buffer system contained 4.1% sucrose and 0.02% polysorbate 20, and the pH was 6.0. The buffer system mainly considers a histidine-histidine hydrochloride buffer system, and the pH values (A60 and CP60) with the best stability of two buffer systems of acetic acid-sodium acetate and citric acid-sodium citrate are used as a control buffer system. Stabilizers include sucrose and sorbitol; the surfactant in this round was tentatively 0.02% polysorbate 20.

In the research, an auxiliary material system is designed and screened through a single-factor test, and the influence of different buffer solution types, sodium chloride and stabilizer types on the stability of the protein is inspected. HLX11 adjuvant initial screening study candidate prescription information is shown in Table 8.

TABLE 8 formulation composition of HLX11 formulation adjuvants for first round screening study candidate formula

The preparation method comprises the following steps: HLX11 protein obtained in the examples is purified by three-step chromatography, and auxiliary materials and protein concentration are added after ultrafiltration liquid change to prepare the formula shown in Table 8. Respectively filtering the samples by using a disposable sterile filter with the diameter of 0.22 mu m in a biological safety cabinet, aseptically subpackaging 1.0mL of the samples into 2mL of sterile penicillin bottles, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. Placing the above samples in a constant temperature and humidity chamber at 40 deg.C (75% RH) for 4 weeks, respectively, sampling at 0, 2 and 4 weeks, and detecting and analyzing to obtain appearance and protein content (A)₂₈₀) pH, molecular isomer (SEC-HPLC), protein particle size and PdI (DLS), charge isomer (CEX-HPLC), etc. HLX11 formulation adjuvant screening study examination conditions are detailed in Table 9.

TABLE 9 HLX11 Accessory first round screening study examination conditions

The results of the initial screening study of HLX11 adjuvant are shown in Table 10.

As can be seen from table 10, the addition of NaCl in the formula HSuN resulted in a heavier opalescence of the protein solution, and the opalescence of the formula C60 protein solution was also slightly more pronounced than the solutions under the other two sets of buffer systems. HLX11 pharmaceutical formulation excipients the results of the 4-week purity change comparison are shown in figure 1. Wherein, fig. 3 (a): SEC main peak content variation trend comparison graphs of different prescriptions; fig. 3 (B): SEC fragment content trend comparison graph of different prescriptions; fig. 3 (C): comparing the content variation trend of the CEX main peak of different prescriptions; fig. 3 (D): a CEX acidic peak content variation trend comparison graph of different prescriptions; fig. 3 (E): graph comparing the change of the average particle size of DLS protein of different prescriptions.

As can be seen from the SEC data in table 10 and fig. 3(a) to (B), the SEC main peak content at each position is decreased in comparison with 0 week after 4 weeks of acceleration at 40 ℃, and the decrease in the main peak content is mainly manifested by a significant increase in the fragment content. The main peak antibody levels in the prescribed HASU and HSuN decreased faster than those in the other prescribed formulas, while the main peak antibody levels in the prescribed HSu, HSo, ASu and CSu decreased nearly; the main peak content of the HSo antibody decreased slightly more slowly than HSu.

Combining the CEX data in table 10 and fig. 3(C) - (D), the main peak content in each formula showed a decrease trend after 4 weeks of acceleration at 40 ℃, and the decrease in the main peak content was mainly manifested as a significant increase in the acid peak content, compared with 0 weeks. The decrease in the main peak antibody levels in the prescribed HASU and CSu was faster than the other prescribed, the decrease in the main peak antibody level in the prescribed HSuN was slightly slower, and the decrease in the main peak antibody levels in the prescribed HSu, HSo and ASu was consistent and slowest.

As can be seen from table 10 and fig. 3(E), the addition of NaCl in the formula HSuN also resulted in a significant increase in the protein particle size; compared with the protein particle size under a histidine-histidine hydrochloride buffer system, the protein particle sizes of the formulas ASu and CSu are larger, and the acetic acid-sodium acetate buffer system and the citric acid-sodium citrate buffer system are further proved to be easy to generate polymers.

In conclusion, according to the results of the initial research accelerated stability test of the auxiliary material screening, when the pH value of a 20mmol/L histidine-histidine hydrochloric acid buffer system is 6.0, 3.0% sorbitol is added as a stabilizer, and the protein stability is the best. Therefore, we selected 20mmol/L histidine-histidine hydrochloride buffer system with pH6.0 and 3.0% sorbitol as stabilizer for subsequent surfactant type and content study.

3.2 Secondary screening of adjuvants

In the research, an auxiliary material system is further designed and screened through a single-factor test, and the influence of different types and contents of surfactants is investigated. HLX11 adjuvant screening sub-round study candidate prescription information is shown in Table 11.

TABLE 11 formulation compositions of HLX11 formulation adjuvants for a second round of screening studies

The preparation method comprises the following steps: HLX11 protein obtained in example 1 is purified by three-step chromatography, and is prepared into a formula shown in Table 11 by adding auxiliary materials and adjusting the concentration of protein after ultrafiltration liquid change. Respectively filtering the samples by using a disposable sterile filter with the diameter of 0.22 mu m in a biological safety cabinet, aseptically subpackaging 1.0mL of the samples into 2mL of sterile penicillin bottles, adding a 13mm rubber plug, and rolling a 13mm aluminum-plastic combined cover. Respectively placing the above sub-packaged samples in constant temperature shaking table at 40 deg.C (75% RH) at 200rpm for 4 weeks, respectively sampling at 0, 1, 2 and 4 weeks, and detecting and analyzing to obtain appearance and protein content (A)₂₈₀) pH value, molecular isomer (SEC-HPLC), charge isomer (CEX-HPLC) and protein particle size, PdI (DLS), and the like. The HLX11 formulation adjuvant screening secondary study examination conditions are detailed in table 12.

TABLE 12 HLX11 Accessory selection study examination conditions

The results of the secondary screening study with HLX11 adjuvant are shown in Table 13.

TABLE 13 summary of data from HLX11 formulation adjuvants in a second round of screening studies accelerated stability tests (200 rpm at 40 ℃ C.)

As shown in table 13, each test result showed good protein quality at 0 week, and there was no significant difference between the alternative prescriptions. After 4 weeks of examination at 40 ℃ and 200rpm, the basic physicochemical properties (appearance, protein content and pH value) of each formula were not significantly changed from the average particle size of the protein and PdI.

The SEC data shows that the SEC main peak content of each prescription antibody shows a descending trend, the dimer has no obvious change, and the main peak content is reduced mainly through the obvious increase of the fragment content, wherein the SEC main peaks of the prescription F1 (without polysorbate 20 or 80) and F2 (with 0.02% of polysorbate 20) antibodies are reduced the least, and the SEC main peaks of the prescription F3 (with 0.05% of polysorbate 20) and F4 (with 0.02% of polysorbate 80) antibodies are reduced slightly quickly.

The CEX data show that the main peak content of each prescription CEX antibody shows a descending trend, the alkali peak has no obvious change, and the main peak content of the CEX antibody is reduced mainly through the obvious increase of the acid peak content, wherein the main peak of the prescription F1 (without polysorbate 20 or 80) antibody is reduced the least, the main peak of the F2 (with 0.02% of polysorbate 20) antibody is reduced slightly quickly, and the main peaks of the F3 (with 0.05% of polysorbate 20) and the F4 (with 0.02% of polysorbate 80) antibody are reduced the fastest. The results of comparing the purity change of HLX11 formulation surfactant screening for 4 weeks are shown in figure 4. Wherein FIG. 4(A) is a graph comparing the change in SEC main peak content; FIG. 4(B) is a graph comparing the change in SEC fragment content; FIG. 4(C) is a graph comparing the change in the content of the main CEX peak; FIG. 4(D) is a graph comparing the change in the content of the CEX acidic peak.

In summary, combining the SEC purity and CEX purity change comparison plots (FIGS. 4(A) - (D)), protein stability was slightly better when 0.02% PS20 was added to the recipe than when 0.02% PS80 was added. When the concentration of PS20 added in the prescription is 0.00%, 0.02% and 0.05% (prescription F1-F3), the stability of protein is not obviously different, and all the alternative prescriptions have good stability. Therefore, the content of PS20 in the formula of the HLX11 preparation can be controlled within the range of 0.20 +/-0.2 mg/mL.

Through the development of a preparation prescription, a buffer system and auxiliary materials which are helpful for the stabilization of HLX11 protein are screened out, and the composition of the HLX11 pharmaceutical preparation is determined as follows: 30mg/mLHLX11 monoclonal antibody, 1.51mg/mL histidine, 2.15mg/mL histidine hydrochloride (the molar concentration of histidine-histidine hydrochloride buffer solution is 20mmol), 30.0mg/mL sorbitol, 0.2mg/mL polysorbate 20 (the volume percentage content of polysorbate 20 is 0.02%), pH value is 6.0.

Example 4 HLX11 formulation vs Perjeta Primary stability control study

According to the description of example 1, the HLX11 protein exchange solution obtained by the final cultivation and purification process was diluted to the final prescription, and using the same inner packaging material and specifications, the HLX11 pharmaceutical formulation and Perjeta (purchased from roche) were separately filtered through a 0.22 μm disposable sterile filter in a biosafety cabinet, aseptically dispensed 1.0mL sample into 2mL sterile vials, added with 13mm plugs, and rolled with 13mm aluminum plastic combined covers.

The formula of the Perjeta original developing agent is as follows: 30mg/mL Pertuzumab, 20mM L-histidine acetate, 120mM sucrose and 0.02% polysorbate 20, pH 6.0.

The examination conditions were 50 ℃ and 1 month, and the samples were taken at 0 week, 1 week, 2 weeks and 4 weeks for examination, including appearance, protein content, osmolality and purity (SEC-HPLC, CEX-HPLC, CE-SDS). The HLX11 pharmaceutical formulations are examined with the Perjeta preliminary stability control test in table 14.

TABLE 14 HLX11 pharmaceutical formulation vs Perjeta preliminary stability control test investigation conditions

The results of the HLX11 vs Perjeta preliminary stability control are detailed in Table 15.

TABLE 15 results of HLX11 pharmaceutical formulations in comparison with Perjeta preliminary stability study

Initial stability studies of HLX11 pharmaceutical formulations the results of accelerated test SEC and CEX for the change in the content of each component are shown in figure 5. Wherein in FIG. 5(A), SEC main peak content trend graph; FIG. 5(B) SEC fragment content trend graph; FIG. 5(C) is a linear fit trend plot of SEC main peak content change; FIG. 5(D) is a linear fit trend plot of SEC fragment content change; FIG. 5E is a CEX main peak content trend graph; FIG. 5(F) is a CEX acidic peak content trend chart; FIG. 5(G) is a line-fit trend plot of the change in CEX main peak content; FIG. 5(H) is a linear fit of the trend plot for the change in CEX acid peak content.

The SEC data in combination table 15 and fig. 5(a) - (D) show that the main peak content of each prescription antibody SEC is in a decreasing trend, the dimer is slightly increased, and the decrease of the main peak content is mainly shown as a significant increase of the fragment content. The linear fitting equations of the SEC main peak content change of HLX11 and Perjeta are that y is-2.25 x +102.2 and y is-2.20 x +102.2, respectively, the main peak descending slope-2.25 of HLX11 and the main peak descending slope-2.2 of Perjeta have small difference, so the SEC main peak descending trend of HLX11 and Perjeta is consistent. The linear fitting equation of the SEC segment content change of HLX11 and Perjeta is that y is 2.05x +2.4 and y is 1.85x +1.95, the SEC segment increase slope 2.05 of HLX11 is greater than the segment increase slope 1.85 of Perjeta, so the SEC segment increase trend of HLX11 is slightly faster than the segment increase trend of Perjeta. However, after 4 weeks at 50 ℃ the SEC fragment increased by 6.4% (HLX11) and 5.5% (Perjeta), respectively, with negligible difference.

The CEX data in table 15 and fig. 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 significantly, and the decrease of the main peak content of CEX is mainly shown by the significant increase of the acid peak content. The linear fitting equations of the HLX11 and the Perjeta CEX main peak content change are that y is-16.39 x +92.9 and y is-17.27 x +90.55, respectively, the main peak descending slope-16.39 of the HLX11 is slightly larger than that of the Perjeta-17.27, and therefore the HLX11CEX main peak descending trend is slightly slower than that of the Perjeta. The linear fitting equations of the CEX acid peak content change of HLX11 and Perjeta are respectively that y is 15.89x +3.55 and y is 18.59x-4.95, the acid peak increase slope of HLX11 is 15.89 which is obviously smaller than that of Perjeta 18.59, and thus the acid peak increase trend of HLX11 is obviously slower than that of Perjeta.

In conclusion, the HLX11 pharmaceutical formulation obtained by the invention has slightly lighter opalescence than the Perjeta preparation, and the osmotic pressure is closer to the human blood osmotic pressure; the SEC purity trend of the HLX11 formulation was consistent with that of the Perjeta formulation; the main peak of HLX11CEX has a descending trend slightly slower than that of Perjeta, and the peak increasing trend of HLX11CEX is obviously weaker than that of Perjeta. Compared with the Perjeta preparation of the original medicine, the HLX11 pharmaceutical preparation is more beneficial to antibody stabilization, and can ensure the effective period of more than four weeks under the storage condition of not higher than 50 ℃.

It will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above disclosure, and equivalents may fall within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shanghai Fuhonghanlin biotechnological GmbH

SHANGHAI FUHONG HANLIN BIOPHARMACY Co.,Ltd.

<120> a pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody

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Claims

1. A pharmaceutical formulation comprising an anti-Her 2 monoclonal antibody, wherein said pharmaceutical formulation comprises: an anti-Her 2 monoclonal antibody, a buffer, a protein protectant, and a surfactant.

2. The pharmaceutical formulation of claim 1, wherein the buffer is: one or more of citric acid-sodium citrate buffer solution, histidine-histidine hydrochloride buffer solution, acetic acid-sodium acetate buffer solution and citric acid-disodium hydrogen phosphate buffer solution, wherein the concentration of the buffer solution is 10-30 mM.

3. The pharmaceutical formulation of claim 2, wherein the buffer is histidine-histidine hydrochloride at a concentration of 20 mM.

4. The pharmaceutical formulation of claim 1, wherein the protein protectant is: one or more of sucrose, trehalose, glycine, mannitol, sorbitol and/or arginine hydrochloride; the mass percentage content is 1% -5%.

5. The pharmaceutical formulation of claim 1, wherein the surfactant is one or more of a polysorbate, polysorbate 20, and/or a poloxamer, and the surfactant content is 0.1mg/mL to 0.4 mg/mL.

6. The pharmaceutical formulation of claim 5, wherein the light chain amino acid sequence of said anti-Her 2 monoclonal antibody is as set forth in SEQ ID NO:1, and the heavy chain amino acid sequence thereof is shown as SEQ ID NO:2, respectively.

7. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation has a pH of 5 to 7.

8. The pharmaceutical formulation of claim 1, wherein said anti-Her 2 monoclonal antibody has a protein concentration of 5mg/ml to 50 mg/ml.

9. The pharmaceutical formulation of any one of claims 1 to 8, wherein the pharmaceutical formulation comprises: 30mg/mL of anti-Her 2 monoclonal antibody, 20mmol/L of histidine-histidine hydrochloride buffer solution, 3% by mass of sorbitol, 0.2mg/mL of polysorbate 20, and the pH value of the solution is 6.0.

10. The pharmaceutical formulation according to any one of claims 1 to 9, wherein the pharmaceutical formulation is a liquid formulation for injection or a lyophilized formulation.