WO2022017401A1 - Anti-her2/pd1 bispecific antibody lyophilized formulation and preparation method therefor - Google Patents

Abstract

An anti-HER2/PD1 bispecific antibody lyophilized formulation and a preparation method therefor. By optimizing a formula of a formulation and a lyophilization process, the defect of instability of a scFv-IgG C-fused bispecific antibody is greatly improved. According to the formula of the formulation and the preparation method, the SEC purity and IEC purity measured by means of a stability evaluation method known in the art, for example, storing for 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, or 36 months at 5±3°C, should meet quality standards.

Description

Anti-HER2/PD1 bispecific antibody freeze-dried preparation and preparation method thereof technical field

The invention belongs to the field of biopharmaceuticals, and in particular relates to a freeze-dried preparation of an anti-HER2/PD1 bispecific antibody and a preparation method thereof.

Background technique

HER2 (human epidermal growth factor receptor2) has receptor tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family. It is only expressed at a low level in a few normal tissues of adults. However, studies have shown that HER2 is overexpressed in a variety of tumors, such as about 30% of breast cancer patients and 16% of gastric cancer patients. Tumor growth and enhanced tumor invasive and metastatic capabilities are important indicators of poor prognosis in such patients.

Human programmed cell death receptor-1 (PD-1) is a type I membrane protein composed of 288 amino acids, the extracellular segment is the Ig variable (V-type) domain responsible for binding ligands, and the intracellular segment is The cytoplasmic tail responsible for binding signal transduction molecules. The PD-1 cytoplasmic tail contains two tyrosine-based signal transduction motifs, ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine switching motif), respectively. . PD-1 is expressed on the surface of activated T lymphocytes, and it interacts with the ligands PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed death receptor-ligand 1). Body 2, programmed cell death-Ligand 2) binding can inhibit the activity of T lymphocytes and related in vivo cellular immune responses. Numerous studies have shown that the interaction of PD-1 and PD-L1 not only maintains the balance of the immune system in the body, but also is the main mechanism that causes PD-L1-positive tumor cells to evade immune surveillance. By blocking the PD-1/PD-L1 signaling pathway, it can activate the immune system and restore the immune killing function of T cells.

Bispecific antibodies (BsAbs) refer to antibody molecules that can simultaneously bind to two (or more) different epitopes. Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: 1) Bispecific antibodies can bind 2 or more different antigen molecules or different epitopes of the same molecule at the same time, and combination drugs often do not. have this effect. 2) Mediate cell-to-cell interactions, bispecific antibodies can bind to two antigens on effector cells and target cells, respectively, to build a bridge between effector cells and target cells, and promote cell-to-cell interactions, such as mediating The killing of tumor cells by immune cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.

PCT patent application WO2020/103629A1 discloses a self-developed anti-HER2/PD1 bispecific antibody, which is an anti-HER2/PD1 bispecific antibody expressed in CHO cells using DNA recombinant technology. It consists of a single-chain antibody with two PD1 ends connected (ScFv-IgG C-terminal fusion). ScFv-IgG C-terminal fusion bispecific antibodies are very easy to cause molecular aggregation and precipitation because the C-terminal ScFv expands and contracts in solution state and presents a respiratory chain state; at the same time, it is easy to expose internal amino acids during expansion and contraction, resulting in oxidation and deamination Occurs, forming charge isomers with poor stability. Therefore, it is necessary to improve the stability through formulation optimization.

SUMMARY OF THE INVENTION

In order to solve the above problems, the purpose of the present invention is to provide a lyophilized preparation of anti-HER2/PD1 bispecific antibody and a preparation method thereof. The lyophilized preparation consists of an anti-HER2/PD1 bispecific antibody, a buffer, a protein protectant, and a surfactant. The preparation method includes the processes of medicinal liquid preparation, medicinal liquid subpackaging, freeze-drying and the like.

In order to realize the purpose of the present invention, the present invention adopts the following technical solutions:

A first aspect of the present invention provides a lyophilized preparation of an anti-HER2/PD1 bispecific antibody, the lyophilized preparation comprising an anti-HER2/PD1 bispecific antibody, a buffer, a protein protectant, and a surfactant.

Wherein, the concentration of the anti-HER2/PD1 bispecific antibody is 5-20 mg/ml, and the anti-HER2/PD1 bispecific antibody comprises the heavy chain shown in SEQ ID NO: 1 and the heavy chain shown in SEQ ID NO: : the light chain shown in 2. Preferably, the concentration of the anti-HER2/PD1 bispecific antibody is 10-15 mg/ml, and more preferably, the concentration of the anti-HER2/PD1 bispecific antibody is 10 mg/ml.

Wherein, the buffer is histidine-histidine hydrochloride buffer, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM. Preferably, the concentration of the histidine-histidine hydrochloride buffer is 10 mM.

Wherein, the protein protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of the trehalose or sucrose is 10-50 mg/ml, and the concentration of the arginine hydrochloride is 25-50 mM. Preferably, the protein protecting agent is trehalose and arginine hydrochloride, the concentration of trehalose is 50 mg/ml, and the concentration of arginine hydrochloride is 50 mM.

Wherein, the surfactant is polysorbate 80 or polysorbate 20, the concentration of polysorbate 80 or polysorbate 20 is 0.1-0.5mg/ml, preferably, the polysorbate 80 or polysorbate 20 at a concentration of 0.2-0.5 mg/ml. Preferably, the surfactant is polysorbate 80, and the concentration of the polysorbate 80 is 0.4±0.05mg/ml.

Wherein, the pH of the freeze-dried preparation is 6.8±0.5.

In another preferred embodiment, the freeze-dried preparation includes:

Anti-HER2/PD1 bispecific antibody at a concentration of 10-15mg/ml;

Histidine-Histidine HCl buffer at a concentration of 10-20 mM;

Trehalose at a concentration of 10-50 mg/ml;

Arginine hydrochloride at a concentration of 25-50 mg/ml;

Polysorbate 80 at a concentration of 0.2-0.5 mg/ml; and

The pH of the lyophilized formulation was 6.8±0.5.

In another preferred embodiment, the freeze-dried preparation does not contain antioxidants.

The second aspect of the present invention provides the preparation method of the freeze-dried preparation, the preparation method includes the processes of liquid medicine preparation, liquid medicine sub-packaging, freeze-drying and the like.

Wherein, the medicinal solution is prepared as a stock solution or a medicinal solution of a preparation. The preparation of the medicinal liquid includes the ultrafiltration, concentration, and liquid exchange process and the dilution process of the protein solution. The ultrafiltration concentration liquid exchange process comprises the following steps: first, adding a protein protective agent to the protein solution to be ultrafiltration concentrated and ultrafiltration concentrated, the protein protective agent is trehalose or sucrose, arginine hydrochloride, so The concentration of described trehalose or sucrose is 10-50mg/ml, preferably 10mg/ml, and the concentration of described arginine hydrochloride is 25-50mM, preferably 50mM; Secondly, the protein solution after the ultrafiltration concentration is carried out. Change the liquid, the liquid change solution comprises a histidine-histidine hydrochloride buffer and a protein protective agent, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM, preferably 10 mM, and the protein The protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of the trehalose or sucrose is 10-50mg/ml, preferably 20mg/ml, the concentration of the arginine hydrochloride is 25-50mM, preferably 50mM. The dilution process is to add an appropriate amount of buffer, protein protective agent and surfactant to the protein solution after the liquid exchange, so that the protein solution reaches the components and dosage of the lyophilized preparation provided in the first aspect of the present invention.

Wherein, the dosage of the medicinal liquid is 5-160mg/bottle, preferably, the dosage of the medicinal liquid is 10-50mg/bottle, more preferably, the dosage of the medicinal liquid is 50mg/ bottle.

Wherein, the freeze-drying includes the steps of pre-freezing, main drying, secondary drying and final drying, the pre-freezing temperature is 5°C, the pre-freezing temperature is -40°C, and the pre-freezing time is not less than 3h ; The main drying temperature is -20--12°C, the time is not less than 21h, and the vacuum degree is 200-250μbar; the secondary drying temperature is -5-15°C, and the time is not less than 12.5h, The vacuum degree is 100-250μbar; the final drying temperature is 30°C, the time is not less than 3.3h, and the vacuum degree is 20-60μbar.

The third aspect of the present invention provides the use of the freeze-dried preparation described in the first aspect of the present invention for preparing a medicament for treating cancer or tumor.

A fourth aspect of the present invention provides a kit comprising:

The lyophilized preparation of an antibody according to the first aspect of the present invention; and

A container for holding the lyophilized formulation.

In another preferred embodiment, the kit further includes instructions for use.

Beneficial effect: The present invention greatly improves the unstable defect of the bispecific antibody of the ScFv-IgG C-terminal fusion class by optimizing the formulation formula and the freeze-drying process. Methods for evaluating stability, such as storage at 5±3°C for 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 36 months, the tested SEC purity and IEC purity should be Comply with quality standards.

Description of drawings

Figure 1 shows the results of SEC purity and IEC purity of protein solutions under different pH conditions.

Figure 2 is the result of the effect of protein protectant on the ultrafiltration concentration exchange medium.

Figure 3 shows the analysis of the DOE results.

Figure 4 shows the optimization results of the lyophilized formula.

Figure 5 is the lyophilization curve of 20191210.

Figure 6 is the lyophilization curve of 20191213.

Figure 7 is the lyophilization curve of 20200114.

Figure 8 is the lyophilization curve of 20200214.

Figure 9 is the lyophilization curve of 20200314.

detailed description

The inventors have found through extensive and in-depth research that a freeze-dried preparation of an anti-HER2/PD1 bispecific antibody fused to the C-terminal of ScFv-IgG and a preparation method thereof are provided. Specifically, the lyophilized preparation comprises a specific concentration of the bispecific antibody, a buffer system (such as histidine-histidine hydrochloride buffer), a protein protectant (such as trehalose or sucrose, arginine hydrochloride) and surfactants (such as polysorbate 80 or polysorbate 20). The freeze-dried preparation of the present invention significantly improves the stability of the anti-HER2/PD1 bispecific antibody fused to the C-terminal of ScFv-IgG, and the storage time of the preparation is effectively prolonged. The freeze-dried preparation product of the invention has small batch difference and stable quality, and is suitable for industrial batch production. The present invention has been completed on this basis.

the term

As used herein, "the lyophilized formulation of the antibody of the present invention", "the lyophilized formulation of the anti-HER2/PD1 bispecific antibody", "the lyophilized formulation of the bispecific antibody of the present invention" and "the lyophilized formulation of the present invention" " can be used interchangeably, and both refer to the lyophilized preparation of the anti-HER2/PD1 bispecific antibody described in the first aspect of the present invention.

As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

As used herein, when used in reference to a specifically recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Antibody lyophilized preparation

The antibody freeze-dried preparation of the present invention mainly includes:

(i) anti-HER2/PD1 bispecific antibody;

(ii) buffer;

(iii) protein protectants; and

(iv) surfactants;

Wherein, the concentration of the anti-HER2/PD1 bispecific antibody is 5-20 mg/ml,

The buffer is histidine-histidine hydrochloride buffer, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM;

Described protein protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of described trehalose or sucrose is 10-50mg/ml, and the concentration of described arginine hydrochloride is 25-50mM;

Described surfactant is polysorbate 80 or polysorbate 20, the concentration of described polysorbate 80 or polysorbate 20 is 0.1-0.5mg/ml, the pH of described freeze-dried preparation is 6.8± 0.5.

It should be understood that the concentration of each component in the antibody lyophilized preparation of the present invention, such as the concentration of bispecific antibody, the concentration of buffer, the concentration of protein protectant or the concentration of surfactant, refers to the same volume as before lyophilization The reconstituted concentration in a solution (eg, water, or buffer).

It should be understood that the pH of the antibody lyophilized formulation of the present invention refers to the pH after reconstitution in the same volume of solution (eg, water, or buffer) as before lyophilization.

Lyophilized formulations are generally more chemically stable than solution formulations, thus increasing half-life. Antibody lyophilized formulations can be reconstituted at different concentrations depending on clinical factors such as route of administration or dosage.

The amount of antibody present in the formulations of the invention is determined by taking into account the desired dosage volume and mode of administration. In the present invention, the concentration of the antibody is 5-20 mg/ml, preferably 10-15 mg/ml, most preferably 10 mg/ml. The present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.

The buffer system used in the formulation of the present invention is a buffer system comprising histidine. In the buffer system, histidine can exist alone or in the following forms, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate and the like. In a preferred embodiment, the histidine in the buffer system is present alone or in the form of histidine hydrochloride.

The protein protectant of the present invention is mainly composed of polyols, wherein "polyols" are substances having multiple hydroxyl groups, and include sugars (reducing sugars and non-reducing sugars), sugar alcohols and sugar acids. "Reducing sugars" are sugars that include hemiacetal groups, which are capable of reducing metal ions or covalently react with lysine and other amino groups in proteins, while "non-reducing sugars" are sugars that do not possess the above-mentioned characteristics of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose, and raffinose. Examples of sugar alcohols include mannitol, xylitol, erythritol, threitol, sorbitol and glycerol. As for sugar acids, L-gluconic acid and its metal salts are included. If the formulation is desired to be freeze-thaw stable, the polyol preferably does not crystallize at freezing temperatures (eg, -20°C) so that it destabilizes the antibody in the formulation.

The amount of polyol used can vary depending on the desired isotonicity of the formulation. The formulations of the present invention are preferably isotonic. The amount of polyol added can also vary depending on the molecular weight of the polyol. Preferred polyols of the present invention are sugar alcohols. In a preferred embodiment of the present invention, the polyol is 10-50 mg/ml trehalose or sucrose; preferably 10-50 mg/ml trehalose; more preferably 50 mg/ml trehalose.

The protein protectant in the present invention also includes arginine hydrochloride. In a preferred embodiment of the present invention, the concentration of arginine hydrochloride is 25-50 mM, more preferably 50 mM. The present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.

The surfactants in the formulations of the present invention are preferably nonionic surfactants such as polysorbates or poloxamers. The amount of surfactant added is such that it reduces aggregation of antibodies in the formulation and/or reduces particle formation in the formulation and/or reduces adsorption. Preferred surfactants of the present invention are polysorbates, such as polysorbate 80 or polysorbate 20, more preferably polysorbate 80. In a preferred embodiment, the concentration of polysorbate 80 is 0.1-0.5 mg/ml, preferably, 0.2-0.5 mg/ml; more preferably, 0.35-0.45 mg/ml. The present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.

The present invention adjusts the pH of the formulation through a buffer system to control the pH in the range of 6.0-7.5, Between 6.6-7.0, 6.7 to 6.9, the present invention includes ranges of values using a combination of any of the above as the upper and/or lower limit. In some preferred embodiments, the formulation pH is 6.0-7.0; more preferably, 6.3-7.3.

It should be understood that, in addition to histidine, the buffer system of the present invention may further include one or more other buffer components, and the pH value of the preparation can be controlled within the above-mentioned range by combining with other buffer components. Suitable other buffer components include citrate, phosphate, acetate (eg, sodium acetate), succinate (eg, sodium succinate), and the like.

In a preferred embodiment of the present invention, the buffer system is histidine-hydrochloric acid, wherein the concentration of histidine is 10-20 mM, more preferably 20 mM. The present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit. In another embodiment, the pH of the formulation is adjusted with a mineral acid such as citric, acetic or phosphoric acid.

After extensive experiments and data screening, the inventor found that when the concentration of histidine in the preparation of the present invention is lower than 10mM, the buffering capacity of the buffer system will be significantly limited. When the concentration of histidine is higher than 20mM, the preparation Stability does not improve.

One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), may be included in the formulations of the present invention, provided they are essential to the formulation The desired characteristics of , are not adversely affected. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include other co-solvents; antioxidants, including ascorbic acid and methionine; integrators, such as EDTA; metals complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; and/or salt-forming counterions.

The formulation of the present invention can be prepared by combining various components in a certain concentration by methods known in the art.

The above features mentioned in the present invention or the features mentioned in the embodiments can be combined arbitrarily. All features disclosed in this specification may be used in combination with any composition, and each feature disclosed in the specification may be replaced by any alternative feature serving the same, equivalent or similar purpose. Therefore, unless otherwise stated, the disclosed features are only general examples of equivalent or similar features.

The protein samples used in the following examples were derived from the anti-HER2/PD1 bispecific antibody a disclosed in PCT patent application WO2020/103629A1, the heavy and light chain amino acid sequences of which are shown below.

Heavy chain amino acid sequence of anti-HER2/PD1 bispecific antibody a (SEQ ID NO: 1)

Light chain amino acid sequence of anti-HER2/PD1 bispecific antibody a (SEQ ID NO: 2)

The detection methods used in the following examples are described as follows:

SEC purity and aggregate detection methods:

Mobile phase: 200 mM phosphate buffer, pH 6.8 ± 0.1. It was filtered through a 0.22 μm filter membrane and degassed by ultrasonic before use. Chromatographic column: TSK G3000SWxl, 7.8×300mm 5μm, TOSOH 08541. High Performance Liquid Chromatograph: Waters Alliance e2695 2489 UV/Vis Detector, Dionex Ultimate 3000 VWD-3400(RS) Detector or other suitable HPLC system with UV detector.

System suitability sample: take the reference substance and dilute the concentration to 5.0mg/ml with mobile phase, centrifuge at 13000rpm for 10min, transfer the supernatant to the injection bottle, and put it into the HPLC sample tray. Test sample: Dilute the test sample with mobile phase to a concentration of 5.0 mg/ml, centrifuge at 13000 rpm for 10 min, take the supernatant and transfer it to a sample bottle, and put it into the HPLC sample tray. Chromatographic conditions: column temperature 25±2℃; sample temperature 10±2℃; detection wavelength UV 280nm; injection volume 20μL; flow rate 0.5ml/min.

The chromatographic software was used for integration, and the peak area percentage of each peak was calculated by the peak area normalization method. Acceptable criteria for system suitability: 6-pin system suitability samples, the resolution of both polymer and monomer are ≥1.5, the retention time RSD of the main peak is less than or equal to 1.0%, the peak area RSD of the main peak is less than or equal to 2.0%, and the asymmetry of the main peak is all ≤2.0, the number of theoretical plates is ≥4000. Report results for the test product: The SEC purity of the sample is reported as the peak area percentage of the main monomer peak, and the aggregate content is the peak area percentage of the polymer peak.

IEC purity testing method:

Mobile phase A: 20 mM phosphate buffer, pH 6.5 ± 0.05. It was filtered through a 0.22 μm filter membrane and degassed by ultrasonic before use. Mobile phase B: 20 mM phosphate buffer + 200 mM sodium chloride, pH 6.5 ± 0.05. It was filtered through a 0.22 μm filter membrane and degassed by ultrasonic before use. Chromatographic column: Propac WCX-10, 4×250mm, Thermo Dionex 054993. High Performance Liquid Chromatograph: Waters Alliance e2695, Dionex Ultimate 3000 series or other suitable HPLC system with UV detector.

System suitability sample: take the reference substance and dilute the concentration with mobile phase to 1.0mg/ml, centrifuge at 13000rpm for 10min, transfer the supernatant to the injection bottle, and put it into the HPLC sample tray. Test sample: dilute the test sample with mobile phase to a concentration of 1.0 mg/ml, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a sample bottle, and put it into the HPLC sample tray. Chromatographic conditions: column temperature 30±2°C; sample temperature 10±2°C; detection wavelength UV 214 nm; injection volume 20 μL; flow rate 1.0 ml/min. The mobile phase gradient is as follows:

Purity analysis: Calculate the peak area percentage of the main peak, acid peak area and base peak area on the sample spectrum by the peak area normalization method. The IEC purity results report the percent area of the main peak.

Unless otherwise specified, the raw material components used in the following examples are all commercially available.

The following examples and experimental examples further illustrate the present invention, but do not limit the present invention in any form.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise specified.

Example 1 Investigation of the effect of pH on stability

In this example, the stability of protein in pH 5.0-7.0 solution was investigated. Prepare 5 batches of samples as shown in Table 1, place them in a refrigerator at 5±3°C for investigation, and take samples at 0, 7, 14, and 21 days to detect SEC purity and IEC purity. The investigation results are shown in Table 2 and Figure 1.

Table 1. Different pH inspection conditions

Table 2. Results of different pH investigations

As can be seen from the results in Figure 1, the IEC purity results show that there is no obvious difference between the samples in each group; the SEC purity results show that the slope of the pH5.0 and pH5.5 The descending trend is significantly greater than the slope under the conditions of pH6.0-7.0 , so the preferred pH range is 6.0-7.0, which indicates that the pH of neutral alkaline is more conducive to protein stability.

Embodiment 2 The investigation of the influence of arginine hydrochloride and trehalose on ultrafiltration concentration and exchange of liquid

In this example, the effects of adding protein protective agents arginine hydrochloride (Arg.HCl) and trehalose (TRE) on the concentration and exchange of ultrafiltration under the conditions of pH 5.0 and pH 6.6 were investigated respectively, wherein the protein concentration range 4-26 mg/ml in 20 mM histidine-histidine hydrochloride buffer (His) and 20 mM acetate buffer (HAc). During the ultrafiltration concentration process, samples were taken at regular intervals to detect protein concentration, SEC purity, and IEC purity. The investigation results are shown in Table 3 and Figure 2.

Table 3. Influence of protein protectant on the stability of ultrafiltration concentrate exchange solution

It can be seen from the results in Fig. 2 that the aggregates tend to increase with the increase of protein concentration in all sample groups. Among them, the sample group under the condition of pH 5.0 has the largest change in slope and therefore the worst result. In the case of pH 6.6, the result of the sample group with arginine hydrochloride added is better than that of the sample group without arginine hydrochloride, and there is no significant difference between 25mM and 50mM of arginine hydrochloride added, so arginine hydrochloride The concentration in the ultrafiltration concentrated exchange medium can be 25-50 mM. In addition, the sample group that added trehalose on the basis of adding arginine hydrochloride showed better results. There was no significant difference between 10mg/ml and 50mg/ml trehalose, so the trehalose concentration could be 10-50mg /ml. Under the protection of arginine hydrochloride and trehalose, the preferred protein concentration of the preparation is 5-20mg/ml, which can be divided into 2ml vials to 20ml vials for freeze-dried preparations, with a volume of 1-8ml, which can cover the dose 5-160mg.

Example 3 Investigation of the effect of arginine hydrochloride in the freeze-drying process

This example investigates the effect of arginine hydrochloride in the freeze-drying process. Three groups of samples were prepared according to Table 4, respectively, and packed into 2ml vials with a volume of 1ml and a dose of 15mg/bottle. According to the freeze-drying process shown in Table 5, the SEC purity was detected before pre-freezing, after pre-freezing and after freeze-drying. The results are shown in Table 6.

Table 4, arginine hydrochloride investigation conditions

Table 5. Freeze-drying parameters

Table 6, the investigation result of arginine hydrochloride

It can be seen from the results in Table 6 that with the increase of the concentration of arginine hydrochloride, the protective effect on the protein during pre-freezing and freeze-drying also increases, and the preferred concentration of arginine hydrochloride is 50 mM.

Example 4 Investigation on the influence of pH and polysorbate 80 on stability

This example examines the effects of pH and polysorbate 80 on protein stability. The DOE design was used for investigation, the pH range was set to 6.2-7.0, the concentration of polysorbate 80 was set to 0.1-0.5 mg/ml, and the concentrations of other components were shown in Table 7. After the sample is prepared, it is divided into 2ml vials, with a volume of 1ml and a dose of 15mg/bottle. The liquid state is placed at 40±2°C and 5±3°C, respectively. Samples are taken at 0, 5, 10, and 15 days to detect the purity of SEC and IEC. , Insoluble particles. The investigation results are shown in Table 8 and Figure 3.

Table 7. DOE design table

Table 8. DOE results

DOE model analysis was performed on the SEC purity slope at 5±3°C, the IEC purity slope at 40±2°C, 2 μm particles, and 10 μm particles, and the results are shown in FIG. 3 . DOE adopts the central composite design-effect surface method, and the model is fitted to a continuous surface, so the set experimental parameter range can be properly extrapolated. It can be seen from the results in Fig. 3 that the preferred pH range is 6.3-7.3, and the preferred concentration range of polysorbate 80 is 0.1-0.5 mg/ml.

Example 5 Optimization of lyophilized formula

In this example, the effects of protein concentration, histidine-histidine hydrochloride buffer, and polysorbate 80 on protein were investigated under freeze-drying conditions. Five groups of samples were prepared according to Table 9, with a protein concentration of 10-15 mg/ml, a histidine-histidine hydrochloride buffer concentration of 10-20 mM, and a polysorbate 80 concentration of 0.2-0.4 mg/ml. After aliquoting 1ml volume in 2ml vials (dose 10mg/vial), freeze-dried, placed at 40±2°C, and sampled at 0, 7, 14, 21, and 28 days to detect SEC purity and IEC purity. The investigation results are shown in Table 10 and Figure 4 .

Table 9, protein concentration, histidine-histidine hydrochloride buffer, polysorbate 80 investigation conditions

Table 10, protein concentration, histidine-histidine hydrochloride buffer, polysorbate 80 investigation results

It can be seen from the results in Figure 4 that the SEC purity and IEC purity of each sample group have similar trends, so it can be judged that the protein concentration is 10-15 mg/ml, the histidine-histidine hydrochloride buffer concentration is 10-20 mM, and the polysorbate 80 Concentrations of 0.2-0.4mg/ml are in line with the formulation requirements.

Embodiment 6 medicinal liquid preparation

In the preparation process of the stock solution with batch number P2004, the protein solution (initial concentration of 2.06 mg/ml) to be concentrated and exchanged by ultrafiltration was added with an appropriate amount of trehalose stock solution (containing 10 mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 363mg/ml trehalose, pH 6.8±0.1) to make the trehalose concentration in the solution 10mg/ml, use an ultrafiltration instrument to carry out ultrafiltration and concentrate to a protein concentration of about 15mg/ml, and carry out ultrafiltration to change the liquid , the exchange solution is 10mM histidine-histidine hydrochloride buffer, 20mg/ml trehalose, 50mM arginine hydrochloride, pH 6.8±0.1. After 7 cycles of liquid change, add an appropriate amount of trehalose stock solution (containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 363mg/ml trehalose, pH 6.8±0.1) to make the trehalose concentration in the solution. to 50mg/ml, then add an appropriate amount of polysorbate 80 stock solution (containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 50mg/ml trehalose, 20mg/ml polysorbate 80, pH 6.8 ±0.1) Bring the polysorbate 80 concentration in solution to 0.4-0.5 mg/ml. Then use preparation excipient solution (containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 50mg/ml trehalose, 0.4mg/ml polysorbate 80, pH6.8±0.1) to dilute to protein concentration 10.0-10.5mg/ml.

Embodiment 7 freeze-drying process investigation

The freeze-drying process of each group of preparations was investigated according to Table 11, and the freeze-drying curves are shown in Figures 5 to 9, where the unit of temperature is °C, the unit of time is min, and the unit of pressure is μbar.

Table 11. Investigation of freeze-drying process

Through the analysis of freeze-drying results, 20191210, 20200214 and 20200314 have better freeze-drying effect. Therefore, it can be seen that the preferred freeze-drying parameters are: pre-freezing -40℃, not less than 3h; main drying temperature -20--12℃, vacuum 200-250μbar, time not less than 21h; secondary drying temperature -5℃-15 ℃, vacuum 100-250μbar, time not less than 12.5h; final drying temperature 30℃, vacuum 20-60μbar, time not less than 3.3h.

Example 8 Stability investigation

Prepare a batch of preparations, the formula is protein 10mg/ml, L-histidine 1.34mg/ml, histidine hydrochloride 0.28mg/ml, arginine hydrochloride 10.53mg/ml (50mM), trehalose 50mg/ml, poly Sorbitan 80 0.4±0.05mg/ml, pH 6.8±0.5, using 20ml vial, filling volume 5ml, dose 50mg/bottle. The lyophilization process is as shown in Table 12. After lyophilization, the samples were placed at 5±3°C, and samples were taken every week to detect the purity of SEC and IEC. The results are shown in Table 13.

Table 12. Freeze-drying parameters

Table 13. Results of stability investigation

time (week, W) SEC Purity (%) IEC Purity (%) 0 93.49 34.82 1 93.56 35.46 2 93.83 38.23 3 93.86 38.57 4 93.57 38.35 5 93.67 37.23 6 93.60 37.17 7 93.50 \ 8 94.25 36.99

It can be seen from the results in Table 13 that the SEC purity and IEC purity results of the preparation samples within 8 weeks show that the protein is not degraded, so it can be seen that the preparation formula and the medicine produced by the freeze-drying process of the present invention have very good stability.

Example 9 Pilot Sample Stability Investigation

According to the optimized formula protein 10mg/ml, L-histidine 1.34mg/ml, histidine hydrochloride 0.28mg/ml, arginine hydrochloride 10.53mg/ml (50mM), trehalose 50mg/ml, polysorbate 80 0.4 ±0.05mg/ml, pH 6.8±0.5, three batches of pilot samples are produced, the batch is 2000-6000 pieces, 20ml vials are used, the filling volume is 5ml, and the dose is 50mg/bottle. The lyophilization process was consistent with Table 12. After freeze-drying, the samples were placed at 5±3° C. to examine long-term stability, and 25±2° C. to examine accelerated stability. The SEC purity and IEC purity were examined. The results are shown in Table 14.

Table 14. Results of stability investigation of pilot samples

As can be seen from the results in Table 14, the SEC purity and IEC purity results of long-term stability within 6 months showed that the protein did not degrade, and the trend was similar to that of the small test sample developed in Example 8, and the accelerated stability results for 6 months also showed no degradation. Significant changes, so the drug has good stability.

Claims (12)

A lyophilized preparation of an anti-HER2/PD1 bispecific antibody, characterized in that the lyophilized preparation comprises: an anti-HER2/PD1 bispecific antibody, a buffer, a protein protectant, and a surfactant; Wherein, the concentration of the anti-HER2/PD1 bispecific antibody is 5-20 mg/ml, and the anti-HER2/PD1 bispecific antibody comprises the heavy chain shown in SEQ ID NO: 1 and the heavy chain shown in SEQ ID NO: : the light chain shown in 2; The buffer is histidine-histidine hydrochloride buffer, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM; Described protein protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of described trehalose or sucrose is 10-50mg/ml, and the concentration of described arginine hydrochloride is 25-50mM; Described surfactant is polysorbate 80 or polysorbate 20, and the concentration of described polysorbate 80 or polysorbate 20 is 0.1-0.5mg/ml; The pH of the lyophilized formulation was 6.8±0.5. The freeze-dried preparation of claim 1, wherein the concentration of the anti-HER2/PD1 bispecific antibody is 10-15 mg/ml, and the concentration of the polysorbate 80 or polysorbate 20 is 0.2-0.5mg/ml. The freeze-dried preparation of claim 1, wherein the freeze-dried preparation comprises: Anti-HER2/PD1 bispecific antibody at a concentration of 10-15mg/ml; Histidine-Histidine HCl buffer at a concentration of 10-20 mM; Trehalose at a concentration of 10-50 mg/ml; Arginine hydrochloride at a concentration of 25-50 mg/ml; Polysorbate 80 at a concentration of 0.2-0.5 mg/ml; and The pH of the freeze-dried preparation is 6.8±0.5. 4. The freeze-dried preparation according to claim 2 or 3, characterized in that: The concentration of the anti-HER2/PD1 bispecific antibody is 10 mg/ml, The concentration of the histidine-histidine hydrochloride buffer is 10mM, Described protein protective agent is trehalose and arginine hydrochloride, The concentration of described trehalose is 50mg/ml, The concentration of described arginine hydrochloride is 50mM, The surfactant is polysorbate 80, and the concentration of the polysorbate 80 is 0.4±0.05mg/ml. The method for preparing a freeze-dried preparation according to any one of claims 1 to 4, characterized in that it comprises the processes of liquid medicine preparation, liquid medicine sub-packaging and freeze-drying. The preparation method according to claim 5, wherein the medicinal solution is prepared as a stock solution or a medicinal solution of a preparation, and the medicinal solution preparation includes an ultrafiltration, concentration, and liquid exchange process and a dilution process of the protein solution. preparation method as claimed in claim 6, is characterized in that, described ultrafiltration concentration liquid changing process comprises the following steps: First, add a protein protectant to the protein solution to be concentrated by ultrafiltration and concentrate by ultrafiltration, Described protein protective agent is trehalose or sucrose, arginine hydrochloride, The concentration of described trehalose or sucrose is 10-50mg/ml, preferably 10mg/ml, The concentration of described arginine hydrochloride is 25-50mM, preferably 50mM; Secondly, the ultrafiltration-concentrated protein solution is exchanged, and the exchanged solution contains histidine-histidine hydrochloride buffer and a protein protective agent, The concentration of the histidine-histidine hydrochloride buffer is 10-20mM, preferably 10mM, Described protein protective agent is trehalose or sucrose, arginine hydrochloride, The concentration of described trehalose or sucrose is 10-50mg/ml, preferably 20mg/ml, The concentration of the arginine hydrochloride is 25-50 mM, preferably 50 mM. The preparation method according to claim 6, wherein the dilution process is to add an appropriate amount of buffer, protein protective agent and surfactant to the protein solution after the liquid exchange, so that the protein solution reaches the level of claim 1- Components and dosage of the freeze-dried preparation described in any one of 3. The preparation method according to claim 5, characterized in that, the dose of the medicinal liquid is 5-160mg/bottle, preferably, the dose of the medicinal liquid is 10-50mg/bottle, more preferably , the dosage of the medicinal liquid is 50mg/bottle. The preparation method of claim 5, wherein the freeze-drying comprises the steps of pre-freezing, primary drying, secondary drying, and final drying; wherein, The pre-freezing temperature is 5°C, the pre-freezing temperature is -40°C, and the pre-freezing time is not less than 3h; The main drying temperature is -20--12°C, the time is not less than 21h, and the vacuum degree is 200-250μbar; The secondary drying temperature is -5-15°C, the time is not less than 12.5h, and the vacuum degree is 100-250μbar; The final drying temperature is 30°C, the time is not less than 3.3h, and the vacuum degree is 20-60μbar. Use of the freeze-dried preparation according to any one of claims 1 to 4 for the preparation of a medicament for treating cancer or tumor. A test kit, characterized in that the test kit comprises: The antibody lyophilized formulation of any one of claims 1-4; and A container for holding the lyophilized formulation. The kit of claim 12, wherein the kit further comprises instructions for use.

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