CN113769081A

CN113769081A - Stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation

Info

Publication number: CN113769081A
Application number: CN202010521390.2A
Authority: CN
Inventors: 杨泗兴; 杨芳; 黄浩旻; 朱祯平
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2021-12-10
Also published as: WO2021249373A1; CN115666613A

Abstract

The invention provides a stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation, which comprises an anti-human IL-5 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant. The defect that the existing anti-IL-5 monoclonal antibody cannot be prepared into a high-concentration liquid preparation is greatly improved by optimizing the formula. The finished product of the liquid preparation medicine can be stored for at least 36 months at the temperature of 2-8 ℃ and at least 6 months at the temperature of 25 ℃. Therefore, the liquid preparation can provide the stability of the preparation of the anti-human IL-5 monoclonal antibody with high concentration, and has wide industrial application prospect.

Description

Stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation.

Background

Interleukin-5 (IL-5) is a kind of lymphocyte activin secreted by activated T cell, it has biological activity to B cell and eosinophilic granulocyte, it has been shown that, anti-human IL-5 antibody can prevent or reduce some anaphylactic disease (such as asthma) related eosinophilic granulocyte through binding with human IL-5 specifically, and then achieve the goal of treating anaphylactic disease.

Currently, some monoclonal antibody drugs targeting human IL-5 are available on the market, such as Nucala (mepallizumab, mepolizumab, Bosatria) developed by GSK, and Cinqair (reslizumab, Cinqaero, available from pioneer) developed by TEVA. Nucala is a freeze-dried preparation, and is reconstituted with 1.2ml of water for injection to prepare 100mg/ml injection for subcutaneous administration before administration. Cinqair is a liquid preparation, 10ml of protein solution is filled in a 10ml penicillin bottle, the concentration is 10mg/ml, and the administration mode is infusion. The same dosage is 100mg at each time, the Nucala is complex to use and operate clinically, and the administration is inconvenient; cinqair cannot be prepared into a high-concentration protein solution, and can be only administered at a low concentration by intravenous infusion. Therefore, both marketed anti-IL-5 monoclonal antibody drugs have drawbacks in terms of clinical administration.

Disclosure of Invention

In order to solve the above problems, the present invention aims to provide a stable high-concentration liquid preparation of a monoclonal antibody against human IL-5. The liquid preparation consists of an anti-human IL-5 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant, can play a role in stabilizing proteins, ensures that target proteins are stored for at least 36 months at 2-8 ℃ and at least 6 months at 25 ℃, has excellent long-term stability, can be directly administrated by subcutaneous injection in clinical use, and greatly develops clinical accessibility compared with the complicated freeze-dried preparation redissolution or intravenous infusion administration.

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

the invention provides a stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation, which comprises an anti-human IL-5 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant, wherein the concentration of the anti-human IL-5 monoclonal antibody is 50-150mg/ml, and the anti-human IL-5 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 9 and the heavy chain as set forth in SEQ ID NO: 10, or a light chain as shown in figure 10.

Wherein, the concentration of the anti-human IL-5 monoclonal antibody is 60-135mg/ml, preferably, the concentration of the anti-human IL-5 monoclonal antibody is 100 mg/ml.

Wherein the buffer solution is histidine-histidine hydrochloride buffer solution, the concentration of the buffer solution is 10-30mM, preferably, the concentration of the buffer solution is 20-30mM, and more preferably, the concentration of the buffer solution is 20 mM.

The protein protective agent is trehalose or sucrose, the concentration of the trehalose or the sucrose is 60-90mg/ml, preferably, the protein protective agent is trehalose, and the concentration of the trehalose is 80 mg/ml.

Wherein the surfactant is polysorbate 80 or polysorbate 20, the concentration of polysorbate 80 or polysorbate 20 is 0.2-1.0mg/ml, preferably, the surfactant is polysorbate 80, and the concentration of polysorbate 80 is 0.4 mg/ml.

Wherein the pH of the liquid preparation is in the range of 5.2-7.0, and preferably, the pH of the liquid preparation is 5.8.

Wherein the liquid formulation is for subcutaneous administration.

The second aspect of the invention provides the use of the stable high-concentration anti-human IL-5 monoclonal antibody preparation in preparing a medicament for treating eosinophil overexpression mediated diseases.

Wherein, the diseases mediated by the eosinophil over-expression comprise asthma, granulomatosis accompanied with vasculitis multiplex, chronic obstructive pulmonary disease, nasal polyp, allergic dermatitis or hypereosinophilic syndrome. Preferably, the disease mediated by eosinophil overexpression is asthma.

Has the advantages that: the invention greatly improves the defect that the existing anti-human IL-5 monoclonal antibody can not be prepared into a high-concentration liquid preparation by optimizing the formula. The finished product of the liquid preparation medicine can be stored for at least 36 months at the temperature of 2-8 ℃ and at least 6 months at the temperature of 25 ℃, has excellent long-term stability, can be directly administrated by subcutaneous injection during clinical use, and greatly expands the clinical accessibility compared with the complicated freeze-dried preparation redissolution or intravenous infusion administration. Therefore, the liquid preparation can provide the stability of the preparation of the anti-human IL-5 monoclonal antibody with high concentration, and has wide industrial application prospect.

Drawings

Figure 1 is a DOE result analysis of polysorbate 80 and pH.

FIG. 2 shows the results of the concentration analysis of the buffer and the protein protectant.

FIG. 3 shows SEC purity presumption validity period.

FIG. 4 shows the IEC purity estimation validity period.

Detailed Description

The protein samples used in the examples below were derived from the Humanized anti-human IL-5 monoclonal antibody 4-6-Humanized as disclosed in WO2019/120060, whose heavy and light chain amino acid sequences are shown below.

4-6-amino acid sequence of the human heavy chain (SEQ ID NO: 1)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHHINWVRQAPGQGLEWIGYI NPYNDYSRYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDYGNF WYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

4-6-Humanized light chain (SEQ ID NO: 2)

DIQMTQSPSSLSASVGDRVTITCKASQDINSYLSWIQQKPGKAPKTLIHRADRL IDGVPSRFSGSGSGQDFTLTISSLQPEDFATYYCLQYDDFPYTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The detection method used in the following examples is illustrated below:

SEC purity, Polymer detection method:

mobile phase: 200mM phosphate buffer, pH 6.8. + -. 0.1. Filtering with 0.22 μm filter membrane, and degassing with ultrasound. A chromatographic column: TSK G3000SWxl, 7.8X 300mm 5 μm, TOSOH 08541. High performance liquid chromatograph: waters Alliance e 26952489 UV/visible light Detector, Dionex Ultimate 3000 VWD-3400(RS) Detector or other suitable HPLC system equipped with a UV Detector.

System applicability sample: the reference substance is diluted to the concentration of 5.0mg/ml by a mobile phase, centrifuged at 13000rpm for 10min, and the supernatant is taken and transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: diluting the sample concentration to 5.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing into HPLC sample plate. Chromatographic conditions are as follows: the column temperature is 25 +/-2 ℃; the sample temperature is 10 +/-2 ℃; detecting the wavelength UV 280 nm; the injection volume is 20 mu L; the flow rate was 0.5 ml/min.

Integration was performed using chromatography software and peak area normalization was used to calculate the peak area percentage of each peak. Acceptance criteria for system suitability: the separation degree of polymers and monomers of 6-needle system applicability samples is more than or equal to 1.5, the retention time RSD of a 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%, the asymmetry of the main peak is less than or equal to 2.0, and the number of theoretical plates is more than or equal to 4000. The test article reports the results: the SEC purity of a sample is reported as the peak area percentage of the monomer main peak and the polymer content as the peak area percentage of the polymer peak.

The IEC purity detection method comprises the following steps:

mobile phase A: 20mM phosphate buffer, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. Mobile phase B: 20mM phosphate buffer +200mM sodium chloride, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and ultrasonic degassing. A chromatographic column: propac WCX-10, 4X 250mm, Thermo Dionex 054993. High performance liquid chromatograph: waters Alliance e2695, Dionex Ultimate series 3000 or other suitable HPLC system equipped with an ultraviolet detector.

System applicability sample: the reference substance is diluted to 1.0mg/ml by mobile phase, centrifuged for 10min at 13000rpm, and the supernatant is transferred to a sample bottle and placed in an HPLC sample tray. And (3) testing the sample: the sample was diluted to 1.0mg/ml with the mobile phase, centrifuged at 13000rpm for 10min, and the supernatant was transferred to the sample bottle and placed in the HPLC sample tray. Chromatographic conditions are as follows: the column temperature is 30 +/-2 ℃; the sample temperature is 10 +/-2 ℃; the detection wavelength is UV 214 nm; the sample volume is 20 mu L; the flow rate was 1.0 ml/min. The mobile phase gradient was as follows:

purity analysis: and calculating the peak area percentages of the main peak, the acid peak area and the alkali peak area on the sample map by using a peak area normalization method. The IEC purity results are reported as peak area percentages of the main peak.

The starting components used in the following examples are commercially available, unless otherwise noted.

The following examples and experimental examples are intended to further illustrate the present invention, but are not intended to limit the present invention in any way.

Example 1 surfactant concentration and pH Effect

As shown in Table 1, in this example, polysorbate 80 and pH were examined by DOE, and the protein concentration was 100mg/ml, polysorbate 80 was in the range of 0-1mg/ml, pH was in the range of 4-7, trehalose was in the range of 80mg/ml, and histidine was in the range of 20 mM. The investigation indexes are SEC purity, IEC purity and insoluble particles. The examination condition was that the sample was left at a high temperature of 40 ℃ for 5 weeks.

Table 1, polysorbate 80 and pH DOE design

The results are shown in Table 2.

TABLE 2 polysorbate 80 and pH DOE test results

The results in table 2 were subjected to regression analysis to obtain the degradation slope for each formulation, and then the slope was subjected to DOE model analysis using JMP, the results of which are shown in fig. 1. As can be seen from FIG. 1, the pH is preferably in the range of 5.2 to 7.0, and the polysorbate 80 concentration is preferably in the range of 0.2 to 1.0 mg/ml.

Example 2 buffer and protein protectant concentration Effect

This example examines the effect of buffer concentration and protein protectant concentration on the formulation, with a protein concentration of 100mg/ml, histidine buffer and protein protectant trehalose concentration as shown in table 3, polysorbate 80 concentration of 0.4mg/ml, and pH of 5.8. After preparation, the mixture is placed at the high temperature of 40 ℃ for 4 weeks, and the detection indexes are SEC purity, IEC purity and insoluble particles.

TABLE 3 buffer and protein protectant concentrations

Numbering	Histidine concentration	TrehaloseConcentration of
			2-1	10mM	60mg/ml
2-2	20mM	60mg/ml
			2-3	30mM	60mg/ml
2-4	20mM	90mg/ml

The results are shown in Table 4.

TABLE 4 buffer and Protektive concentration results

As can be seen from the results in Table 4 and FIG. 2, the results of the two groups 2-2 and 2-3 have no significant difference, but the SEC purity decrease slope of the 2-1 sample is greater than that of the 2-2 and 2-3, therefore, the preferred content range of histidine is 20-30 mM; 2-2 and 2-4, the results are not obviously different, and the trehalose concentration is 60-90mg/ml in consideration of the principle that the injection is isotonic with the human body.

Example 3 protein concentration Effect

This example examines the effect of different protein concentrations on the formulation. 60 and 100mg/ml protein solutions were examined, respectively, with a histidine concentration of 20mM, a trehalose concentration of 80mg/ml, a polysorbate 80 concentration of 0.4mg/ml, and a pH of 5.8. The SEC purity and the polymer content were determined after shaking at 2-8 ℃ for 96 h. The results are shown in Table 5.

TABLE 5 Effect of different protein concentrations on formulations

As can be seen from the results in table 5, the protection in the liquid formulation did not change significantly for the different protein concentrations.

EXAMPLE 4 Final accelerated stability of liquid formulations

3 batches of protein finished products were tried, with a specification of 100mg/ml, a histidine concentration of 20mM, a trehalose concentration of 80mg/ml, a polysorbate 80 concentration of 0.4mg/ml, and a pH of 5.8. Accelerated stability studies were performed for 6 months at 25 ℃ with the results shown in Table 6.

TABLE 6 accelerated stability results for the finished product

From the results in table 6, it can be seen that the protein still meets the standard requirements for SEC purity, IEC purity, insoluble microparticles after 6 months of protective acceleration of the formulation.

EXAMPLE 5 Long term stability of finished product of liquid formulation

3 batches of protein finished products were tried, with a specification of 100mg/ml, a histidine concentration of 20mM, a trehalose concentration of 80mg/ml, a polysorbate 80 concentration of 0.4mg/ml, and a pH of 5.8. The resulting mixture was left at 2 to 8 ℃ for long-term stability studies, and the results are shown in Table 7.

TABLE 7 Long term stability results for the finished product

As can be seen from the results in table 7, the insoluble particles did not significantly change during the protective long-term stability of the formulation, and the SEC purity and IEC purity are shown in fig. 3 and 4, respectively. As can be seen from the analysis, the SEC purity support preparation expiration date is 66 months and the IEC purity support preparation expiration date is 46 months, as predicted by the ICH guidelines, so that the liquid formulation of the present invention can support an expiration date of at least 36 months for a pharmaceutical product.

EXAMPLE 6 stock accelerated stability of liquid formulations

3 batches of drug stock solutions were prepared with protein concentrations of 127, 135, 127mg/ml, histidine concentration 20mM, trehalose concentration 80mg/ml, polysorbate 80 concentration 0.4mg/ml, pH 5.8, respectively. The stock solutions were placed at 2-8 ℃ for accelerated stability studies, and the results are shown in Table 8.

TABLE 8 stock accelerated stability

As can be seen from the results in table 8, the stability of the stock solution was accelerated over 6 months, and there was no significant change in the SEC purity and IEC purity of the stock solution.

Sequence listing

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Claims

1. A stable high-concentration anti-human IL-5 monoclonal antibody liquid preparation comprises an anti-human IL-5 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant, wherein the concentration of the anti-human IL-5 monoclonal antibody is 50-150mg/ml, and the anti-human IL-5 monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 1 and the heavy chain as set forth in SEQ ID NO: 2, or a light chain as shown in figure 2.

2. The liquid preparation according to claim 1, wherein the concentration of the anti-human IL-5 monoclonal antibody is 60-135mg/ml, preferably the concentration of the anti-human IL-5 monoclonal antibody is 100 mg/ml.

3. The liquid formulation of claim 1, wherein the buffer is histidine-histidine hydrochloride buffer, wherein the concentration of the buffer is 10-30mM, preferably wherein the concentration of the buffer is 20-30mM, and more preferably wherein the concentration of the buffer is 20 mM.

4. The liquid formulation of claim 1, wherein the protein protectant is trehalose or sucrose, and the concentration of trehalose or sucrose is 60-90mg/ml, preferably the protein protectant is trehalose, and the concentration of trehalose is 80 mg/ml.

5. The liquid formulation of claim 1, wherein the surfactant is polysorbate 80 or polysorbate 20, the polysorbate 80 or polysorbate 20 concentration is 0.2-1.0mg/ml, preferably the surfactant is polysorbate 80, and the polysorbate 80 concentration is 0.4 mg/ml.

6. The liquid formulation of claim 1, wherein the liquid formulation has a pH in the range of 5.2 to 7.0, preferably wherein the liquid formulation has a pH of 5.8.

7. The liquid formulation of any one of claims 1-6, wherein said liquid formulation is for subcutaneous administration.

8. Use of a liquid formulation according to any one of claims 1-7 for the manufacture of a medicament for the treatment of a disease mediated by eosinophil overexpression.

9. The use of claim 8, wherein said eosinophil overexpression mediated disease comprises asthma, granulomatous disease associated with polyangiitis, chronic obstructive pulmonary disease, nasal polyps, allergic dermatitis, or hypereosinophilic syndrome.

10. The use of claim 9, wherein said eosinophil overexpression mediated disease is asthma.