CN113797330A

CN113797330A - anti-PD-1 monoclonal antibody liquid preparation

Info

Publication number: CN113797330A
Application number: CN202010527399.4A
Authority: CN
Inventors: 杨泗兴; 廖敏; 黄浩旻; 朱祯平
Original assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Current assignee: Sunshine Guojian Pharmaceutical Shanghai Co Ltd
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2021-12-17
Also published as: CN115803055A; WO2021249551A1

Abstract

The invention provides an anti-PD-1 monoclonal antibody liquid preparation, which comprises an anti-PD-1 monoclonal antibody, a buffer solution, a protein protective agent and a surfactant. The liquid preparation can protect the anti-PD-1 monoclonal antibody and improve the stability of the IgG4 type anti-PD-1 monoclonal antibody. The liquid preparation is stored at 2-8 deg.C for at least 60 months, and at 25 deg.C for at least 6 months, and is suitable for clinical application as injection.

Description

anti-PD-1 monoclonal antibody liquid preparation

Technical Field

The invention relates to the field of antibody pharmaceutical preparations, in particular to an anti-PD-1 monoclonal antibody liquid preparation.

Background

Programmed death receptor 1(PD-1) is an important immunosuppressive molecule, and its ligand is programmed death receptor-ligand 1 (PD-L1). The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, promoting proliferation of antigen-specific T cells. One way for tumor cells to escape T cell destruction as antigens is to generate PD-L1 on the surface of the tumor cells, and after PD-1 on the surface of T cells of immune cells recognizes PD-L1, the tumor cells can transmit inhibition signals, and the T cells can not find the tumor cells and send attack signals to the tumor cells. The action mechanism of PD-1 immunotherapy is to design specific protein antibodies against PD-1, prevent the recognition process of PD-1 and PD-L1, partially restore T cell function, thereby enabling T cells to kill tumor cells.

Since anti-PD-1 antibodies only need to bind to PD-1 and do not require ADCC, anti-PD-1 monoclonal antibody molecules are mostly designed as IgG4 type antibodies, such as Keytruda and Opdivo. IgG 4-type antibodies are less stable and more prone to aggregation and insoluble particles, and therefore require good formulation to protect.

Disclosure of Invention

Aiming at the problem of poor stability of the IgG4 type anti-PD-1 monoclonal antibody, the invention aims to provide a stable IgG4 type anti-PD-1 monoclonal antibody liquid preparation. The invention also aims to provide the application of the liquid preparation in preparing a medicament for treating diseases related to PD-1 signaling pathway; and a method for producing the liquid preparation.

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

in a first aspect of the present invention, there is provided an anti-PD-1 monoclonal antibody liquid formulation comprising: an anti-PD-1 monoclonal antibody with the concentration of 20-40mg/ml, a buffer solution with the concentration of 10-30mM, a protein protective agent with the concentration of 40-100mg/ml and a surfactant with the concentration of 0.05-1 mg/ml;

wherein, the anti-human PD-1 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; the buffer solution is selected from an acetate buffer solution, a citrate buffer solution, a histidine-histidine hydrochloride buffer solution or a succinate buffer solution; the protein protective agent is selected from trehalose, sucrose, mannitol, sorbitol, sodium chloride, glucose or fructose; the surfactant is selected from polysorbate 20, polysorbate 80 or poloxamer 188; the pH of the liquid formulation is 5.0-7.0.

In a preferred embodiment, the antibody is an IgG4 class antibody.

In a preferred embodiment, the anti-PD-1 monoclonal antibody is present at a concentration of 25 mg/ml.

In a preferred embodiment, the buffer is a histidine-histidine hydrochloride buffer or an acetate buffer, the buffer concentration being 10 mM.

In a preferred embodiment, the protein protective agent is trehalose, sucrose or mannitol, the concentration of the protein protective agent is 40-95mg/ml, and preferably, the protein protective agent is trehalose, sucrose or mannitol with the concentration of 80-95 mg/ml.

In a preferred embodiment, the surfactant is polysorbate 20 or polysorbate 80 and the surfactant concentration is 0.1 mg/ml.

In a preferred embodiment, the pH of the liquid formulation is from 5.0 to 6.0.

In a preferred embodiment, the formulation has one or two characteristics selected from the group consisting of:

(a) the liquid preparation is stored at 2-8 ℃ for at least 60 months;

(b) the liquid formulation is stored at 25 ℃ for at least 6 months.

In a preferred embodiment, the liquid formulation is for administration by injection.

In a preferred embodiment, the liquid formulation is for intravenous, intramuscular, intraperitoneal or subcutaneous injection administration.

The second aspect of the invention provides the application of the anti-PD-1 monoclonal antibody liquid preparation in preparing a medicament for treating diseases related to PD-1 signaling pathway.

In a preferred embodiment, the disease associated with the PD-1 signaling pathway includes cancer or an autoimmune disease.

The third aspect of the present invention provides a method for preparing the anti-PD-1 monoclonal antibody liquid preparation, which comprises the following steps: (a) mixing an anti-PD-1 monoclonal antibody with the concentration of 20-40mg/ml, a buffer solution with the concentration of 10-30mM, a protein protective agent with the concentration of 40-100mg/ml and a surfactant with the concentration of 0.05-1 mg/ml; (b) adjusting the pH of the mixed mixture to 5.0-7.0;

In a preferred embodiment, the anti-PD-1 monoclonal antibody has a concentration of 25mg/ml, the buffer has a concentration of 10mM, the protein protective agent has a concentration of 80mg/ml to 95mg/ml, the surfactant has a concentration of 0.1mg/ml, and the pH of the liquid formulation is 5.0 to 6.0.

The invention has the beneficial effects that: the anti-PD-1 monoclonal antibody liquid preparation can protect the anti-PD-1 monoclonal antibody and improve the stability of the existing IgG4 type anti-PD-1 monoclonal antibody. The preparation can be stored at 2-8 deg.C for at least 60 months, and at 25 deg.C for at least 6 months, has excellent long-term stability, and is suitable for clinical application as injection.

Drawings

FIG. 1: long-term stability SEC purity and IEC purity detection map

Detailed Description

The following examples are intended to further illustrate the invention and should not be construed as limiting the invention

In the present invention, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies comprised in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically a mixture of different antibodies with epitopes against different antigens), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins.

In the present invention, the term "humanized" means that the CDRs are derived from an antibody of a non-human species (preferably a mouse), and the remaining part of the antibody molecule (including the framework and constant regions) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.

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 ultrasonic degassing. 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 3000VWD-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 the 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: diluting the sample concentration to 1.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing into an 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 injection 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 a main peak, an acid peak area and an 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.

Detailed descriptions of conventional or state-of-the-art methods are not included in the examples below. Such methods are well known to those of ordinary skill in the art and are described in numerous publications.

The proteins in the following examples are referred to as anti-PD-1 antibodies unless otherwise specified.

The anti-PD-1 antibody used in the following examples is a recombinant anti-PD-1 humanized monoclonal antibody derived from monoclonal antibody mAb1-25-humanized disclosed in WO2018137576A1 (application No. PCT/CN 2018/073575). The antibody comprises two identical light chains and two identical heavy chains, and is a recombinant humanized IgG4 monoclonal antibody expressed in CHO cells by adopting a DNA recombination technology. The heavy chain and light chain variable regions of the anti-PD-1 murine antibody are humanized and then respectively connected with the constant regions of the heavy chain and kappa light chain of human IgG4, so that the antibody can be specifically combined with human PD-1. The amino acid sequence of the heavy chain is shown as SEQ ID:1, and the amino acid sequence of the light chain is shown as SEQ ID: 2.

Heavy chain amino acid sequence of mAb1-25-humanized SEQ ID NO: 1

EVKLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWVATISGGGRYTYYPDTVKGRFTISRDNAKNSHYLQMNSLRAEDTAVYFCASPYGGYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Light chain amino acid sequence of mAb1-25-humanized SEQ ID NO: 2

EIVLTQSPATLSLSPGERATLSCRASQSISNFLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNSWPHTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In the following examples, the histidine buffer system refers to histidine-histidine hydrochloride buffer, the acetate buffer system refers to acetate buffer, the citrate buffer system refers to citrate buffer, and the succinate buffer system refers to succinate buffer.

The reagents and starting materials used in the following examples are commercially available unless otherwise specified.

Example 1 Effect of pH on formulations

5 kinds of buffer solutions with different pH values in the table 1 are prepared, and the protein and the auxiliary materials are prepared to obtain the solution of the formula in the table. Respectively sterilizing and filtering through 0.22 mu m filter membrane, subpackaging 1 ml/bottle into 2ml penicillin bottles, plugging, capping to obtain candidate prescription samples, placing the candidate prescription samples in a stability test box at 40 +/-2 ℃, sampling and inspecting at 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks, and inspecting the indexes of SEC purity and IEC purity.

Table 1pH investigation

The results are shown in Table 2, which shows that the SEC purity of the protein in each solution decreases faster with increasing pH, with a better range of pH 4.5-6.0. The change trend of IEC purity indicates that the prescriptions 1-2, 1-3 and 1-4 are superior to the prescriptions 1-1 and 1-5, and indicates that pH5.0-6.0 is beneficial to IEC purity. The comprehensive results show that the pH value is 5.0-6.0, and the protein SEC purity and the IEC purity are better.

Table 2 pH examination results

EXAMPLE 2 Effect of buffer System on formulation

4 different buffer system solutions in the table 3 are prepared, and the protein and the auxiliary materials are prepared to obtain the target prescription solution. Respectively sterilizing and filtering through 0.22 mu m filter membrane, subpackaging 1 ml/bottle into 2ml penicillin bottles, plugging, capping to obtain candidate prescription samples, placing the candidate prescription samples in a stability test box at 40 +/-2 ℃, sampling and inspecting at 1 week, 2 weeks, 4 weeks, 6 weeks and 8 weeks, and inspecting the indexes of SEC purity and IEC purity.

TABLE 3 buffer System screening prescription information

Examining the stability of each prescription at 40 +/-2 ℃, wherein the SEC purity and IEC purity change trends show that the prescription 2-1 and the prescription 2-2 are superior to the prescription 2-3 and the prescription 2-4; the variation trend of other detection items has no obvious difference among all the directions. From these results, it is found that the acetate buffer system and the histidine-histidine hydrochloride buffer system are superior.

TABLE 4 buffer system examination results

Example 3 protein protectant and surfactant effects on formulation

A histidine-histidine hydrochloride buffer system in table 5 was prepared, and the target prescription solution was prepared from the protein and the excipients. Respectively sterilizing and filtering through 0.22 mu m filter membrane, subpackaging 1 ml/bottle into 2ml penicillin bottles, plugging, capping to obtain candidate prescription samples, placing the candidate prescription samples in a stability test box at 40 +/-2 ℃, sampling and inspecting at 1 week, 2 weeks, 4 weeks and 8 weeks, wherein the inspection indexes are SEC purity, IEC purity and insoluble particles.

TABLE 5 protein protectant and surfactant species screening prescription information

The stability, insoluble particle and SEC purity of each prescription are inspected under the condition of 40 +/-2 ℃, and the result further shows that the stability can be effectively improved by adding the saccharide protective agent and the polysorbate in the prescription; the variation trend of other detection items has no obvious difference among all the directions. Therefore, trehalose, sucrose and mannitol can be used as protein protective agents, and polysorbate 80 and polysorbate 20 can be used as surfactants to play a role in solubilization and reduce insoluble particles.

TABLE 6 protein protectant and surfactant Studies

Example 4 Effect of formulation adjuvant dosage on formulation

Preparing each formula solution in the table 7, respectively adding the protein into corresponding auxiliary materials, and adjusting the protein concentration to 25mg/ml by using the additionally prepared auxiliary material solution to obtain the target formula solution. Sterilizing and filtering with 0.22 μm filter membrane, subpackaging 4.3 ml/bottle into 10ml penicillin bottles, capping to obtain each prescription sample, and performing stability inspection on each candidate prescription at 40 + -2 deg.C for 2 months with inspection indexes of SEC purity and IEC purity.

Table 7 recipe information for finding out the amount of excipients

As can be seen from the results in Table 8, there was no significant difference in the trend of each set of recipes. The results of multiple tests show that the dosage of the protein protective agent is preferably 40-100mg/ml, and the dosage of the surfactant is preferably 0.05-1mg/ml, and preferably 0.1 mg/ml.

Table 8 examination of the amount of auxiliary materials

Example 5 accelerated stability test

An anti-PD-1 monoclonal antibody liquid preparation is prepared according to the following preparation formula: 25mg/ml anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer solution, 95mg/ml trehalose, 0.1mg/ml polysorbate 80, pH5.5 +/-0.5. Preparing 3 batches of medicines, placing at 25 +/-2 ℃ for 6 months, sampling at 0, 1, 2, 3 and 6 months to detect the SEC purity and the IEC purity, wherein the results are shown in table 9, and the results show that the medicine purity meets the standard requirements after accelerating for 6 months.

TABLE 9 accelerated stability results

Example 6 Long term stability Studies

An anti-PD-1 monoclonal antibody liquid preparation is prepared according to the following preparation formula: 25mg/ml anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer solution, 95mg/ml trehalose, 0.1mg/ml polysorbate 80, pH5.5 +/-0.5. Batches of 3 were prepared, placed at 5 ± 3 ℃ for long-term stability, sampled at the point of arrival for SEC purity, IEC purity and insoluble particles, and the results are shown in table 10.

TABLE 10 Long term stability results

Analysis of results as shown in fig. 1, it can be seen from fig. 1 that the estimated validity period of the long-term stability SEC purity of the anti-PD-1 monoclonal antibody preparation of the present invention is 167 months, IEC purity does not decrease during long-term stabilization, and insoluble microparticle data does not change significantly, thus supporting a validity period of 60 months.

Sequence listing

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Claims

1. An anti-PD-1 monoclonal antibody liquid formulation, comprising:

an anti-PD-1 monoclonal antibody with the concentration of 20-40mg/ml, a buffer solution with the concentration of 10-30mM, a protein protective agent with the concentration of 40-100mg/ml and a surfactant with the concentration of 0.05-1 mg/ml;

wherein, the anti-human PD-1 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;

the buffer solution is selected from an acetate buffer solution, a citrate buffer solution, a histidine-histidine hydrochloride buffer solution or a succinate buffer solution;

the protein protective agent is selected from trehalose, sucrose, mannitol, sorbitol, sodium chloride, glucose or fructose;

the surfactant is selected from polysorbate 20, polysorbate 80 or poloxamer 188;

the pH of the liquid formulation is 5.0-7.0.

2. The liquid formulation of claim 1, wherein the anti-PD-1 monoclonal antibody is present at a concentration of 25 mg/ml.

3. The liquid formulation of claim 1, wherein the buffer is a histidine-histidine hydrochloride buffer or an acetate buffer, and the buffer concentration is 10 mM.

4. The liquid formulation of claim 1, wherein the protein protectant is trehalose, sucrose or mannitol, the concentration of the protein protectant is 40-95mg/ml, preferably the protein protectant is trehalose, sucrose or mannitol at a concentration of 80-95 mg/ml.

5. The liquid formulation of claim 1, wherein the surfactant is polysorbate 20 or polysorbate 80, and the surfactant concentration is 0.1 mg/ml.

6. The liquid formulation of claim 1, wherein the liquid formulation has a pH of 5.0 to 6.0.

7. The formulation of any one of claims 1 to 6, wherein the formulation has one or two characteristics selected from the group consisting of:

(a) the liquid preparation is stored at 2-8 ℃ for at least 60 months;

(b) the liquid formulation is stored at 25 ℃ for at least 6 months.

8. The liquid formulation of claim 7, wherein the liquid formulation is for administration by injection.

9. Use of a liquid formulation according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment of a disease associated with the PD-1 signalling pathway.

10. The use of claim 9, wherein the disease associated with the PD-1 signaling pathway comprises cancer or an autoimmune disease.