CN115803055A

CN115803055A - anti-PD-1 monoclonal antibody liquid preparation

Info

Publication number: CN115803055A
Application number: CN202180041904.7A
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: 2021-06-11
Publication date: 2023-03-14
Also published as: CN113797330A; WO2021249551A1

Abstract

An anti-PD-1 monoclonal antibody liquid preparation is provided, 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, inhibition signals can be transmitted, and the T cells cannot find the tumor cells and send attack signals to the tumor cells. The mechanism of action of PD-1 immunotherapy is to design specific protein antibodies against PD-1, preventing the recognition process of PD-1 and PD-L1, partially restoring T cell function, thereby allowing 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 anti-PD-1 monoclonal antibody, the invention aims to provide a stable IgG4 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 or a histidine-histidine hydrochloride buffer solution; the protein protective agent is selected from trehalose, sucrose or mannitol; the surfactant is selected from polysorbate 20 or polysorbate 80; the pH of the liquid preparation 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 25mg/ml.

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

In another preferred embodiment, the buffer is histidine-histidine hydrochloride buffer, and the concentration of the buffer is 10mM.

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

In another preferred example, the surfactant is polysorbate 80, and the surfactant concentration is 0.1mg/ml.

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

In another preferred embodiment, the liquid formulation is free of antioxidants (e.g., methionine).

In another preferred embodiment, the formulation comprises:

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 0.1 mg/ml;

the buffer solution is histidine-histidine hydrochloride buffer solution;

the protein protective agent is selected from trehalose, sucrose or mannitol;

the surfactant is polysorbate 80;

the pH of the liquid preparation is 5.0-6.0.

In a preferred embodiment, the formulation has one or more 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 another preferred embodiment, the liquid preparation is stored at 25 +/-2 ℃ for 6 months, and the SEC purity is more than or equal to 97%, preferably more than or equal to 98%, and more preferably more than or equal to 98.5%; and/or the IEC purity is not less than 40%, preferably not less than 42%, more preferably not less than 44%.

In another preferred embodiment, the liquid preparation is stored at 5 +/-3 ℃ for 24 months, and the SEC purity is more than or equal to 97 percent, preferably more than or equal to 98 percent, and more preferably more than or equal to 98.5 percent; and/or the IEC purity is not less than 40%, preferably not less than 42%, more preferably not less than 44% or not less than 46%.

In another preferred embodiment, the liquid formulation is stored at 25 + -2 deg.C for 6 months, and the SEC purity varies by less than or equal to 2%, preferably less than or equal to 1%, more preferably less than or equal to 0.5%, compared to the liquid formulation at day 0; the IEC purity variation range is less than or equal to 6 percent, preferably less than or equal to 5 percent, and more preferably less than or equal to 4 percent.

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 0.1 mg/ml; (b) adjusting the pH of the mixed mixture to 5.0-6.0;

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 or a histidine-histidine hydrochloride buffer solution; the protein protective agent is selected from trehalose, sucrose or mannitol; the surfactant is selected from polysorbate 20 or polysorbate 80; the pH of the liquid preparation is 5.0-6.0.

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 liquid formulation has a pH of 5.0 to 6.0.

A fourth aspect of the invention provides a kit comprising:

a liquid preparation of the antibody according to the first aspect of the invention; and

a container for holding the liquid formulation.

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

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 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 (1A) and IEC purity (1B) are examined.

Detailed Description

The present inventors have extensively and intensively studied and found that a liquid formulation comprising an anti-PD-1 antibody, a buffer system (e.g., an acetate buffer system or a histidine buffer system) at a specific concentration, a protein protecting agent (e.g., trehalose or sucrose), and a surfactant (e.g., polysorbate 80 or polysorbate 20) not only maintains excellent stability in terms of appearance, protein concentration, turbidity, purity, etc., but also remarkably improves chemical stability of the formulation and remarkably reduces insoluble particles in the formulation, at a pH of 5.0 to 6.0; the storage time of the preparation is effectively prolonged. The present invention has been completed on the basis of this finding.

Term(s) for

As used herein, the term "liquid formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective without the inclusion of other ingredients that would be unacceptably toxic to a subject to whom the formulation is administered. The subject includes a mammal, preferably a human.

As used herein, the term "antibody stability" refers to an antibody that substantially retains its physical and/or chemical stability and/or biological activity upon storage. The shelf life is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring antibody stability are known in the art.

Stability can be measured at a selected temperature for a selected time. Preferably, the formulation is stable for at least 1 month at room temperature or 30 ℃ to 40 ℃ and/or for at least 2 years at about 2-8 ℃.

The antibody in the formulation retains its physical stability if it exhibits substantially no evidence of significant aggregation, precipitation and/or denaturation as determined by visual inspection of color and/or clarity, or by UV light scattering or by size exclusion chromatography.

An antibody retains its chemical stability in a formulation if it is chemically stable at a particular time such that the antibody is considered to still retain the biological activity defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibodies. Chemical changes may involve size changes (e.g., shearing), for example, as may be assessed by using size exclusion chromatography, SDS-PAGE and/or matrix-assisted laser-resolved ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation), which can be assessed, for example, by ion exchange chromatography.

An antibody in a formulation retains its biological activity in the formulation if the antibody has biological activity for its intended use. For example, an antibody in the formulation is considered to retain its biological activity (e.g., as determined by an antigen binding assay) if its biological activity is within about 70% to 130% (within the error of the assay) of the biological activity exhibited when the formulation was prepared.

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 region and the constant region) is derived from a human antibody. In addition, framework region residues may be altered to maintain binding affinity.

Liquid preparation of antibodies

The antibody liquid preparation mainly comprises:

(i) 20-40mg/ml of an anti-PD-1 monoclonal antibody;

(ii) A buffer solution with the concentration of 10-30 mM;

(iii) Protein protectant with concentration of 40-100 mg/ml; and

(iv) A surfactant.

The amount of antibody present in the formulation of the invention is determined by taking into account the required dosage volume and mode of administration. In the present invention, the concentration of the antibody is 20 to 40mg/ml, preferably 25 to 30mg/ml, and most preferably 25mg/ml. The present invention includes ranges of values that use any combination of the above values as the upper and/or lower limits.

The buffer system used in the formulation of the present invention may be an acetate buffer system or a buffer system comprising histidine. Histidine-containing buffer systems are preferred, in which histidine may be present alone or in the form of, for example, histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate, etc. In a preferred embodiment, histidine is present in the buffer system either alone or as histidine hydrochloride.

The protein protectant of the present invention consists essentially of a polyol, where "polyol" is a substance having multiple hydroxyl groups and includes sugars (reducing and non-reducing sugars), sugar alcohols, and sugar acids. "reducing sugars" are sugars that include hemiacetal groups that are capable of reducing metal ions or covalently reacting with lysine and other amino groups in proteins, while "non-reducing sugars" are sugars that do not have the above-described 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 sugar acids, L-gluconic acid and its metal salts are included. If it is desired that the formulation be freeze-thaw stable, the polyol is preferably non-crystallizing at freezing temperatures (e.g., -20 ℃) so that it destabilizes the antibodies in the formulation.

The amount of polyol used may vary depending on the desired isotonicity of the formulation. The formulations of the present invention are preferably isotonic. The amount of polyol added may 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 invention, the polyol is trehalose, sucrose or mannitol, the concentration of trehalose or sucrose is preferably 80-95mg/ml and the concentration of mannitol is preferably 40mg/ml. The present invention includes ranges of values that use any combination of the above values as upper and/or lower limits.

The surfactant in the formulation of the invention is preferably a non-ionic surfactant, such as a polysorbate or poloxamer. The amount of surfactant added is such that it reduces aggregation of the antibody in the formulation and/or reduces formation of particles in the formulation and/or reduces adsorption. A preferred surfactant of the invention is a polysorbate, such as polysorbate 80 or polysorbate 20, more preferably polysorbate 80. In a preferred embodiment, polysorbate 80 is present at a concentration of 0.1mg/ml. The present invention includes ranges of values that use any combination of the above values as the upper and/or lower limits.

The present invention includes ranges of values using combinations of any of the above as upper and/or lower limits, by adjusting the pH of the formulation via a buffer system to control the pH in the range of 5.0-6.0, in certain embodiments between 5.0 and 6.0, 5.1 and 5.9, 5.2 and 5.8, 5.3 and 5.7, and 5.4 and 5.6. In some preferred embodiments, the formulation pH is 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

It will be appreciated that the buffer system of the present invention may further comprise one or more other buffer components in addition to histidine, the pH of the formulation being controlled within the above ranges by combination with the other buffer components. Suitable additional buffer components include citrate, phosphate, acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), and the like.

In a preferred embodiment of the invention, the buffer system is histidine-hydrochloric acid, wherein the histidine concentration is in the range of 10-30mM, preferably 10-25mM, more preferably 10mM. The present invention includes ranges of values that use any combination of the above values as upper and/or lower limits. In another embodiment, the pH of the formulation is adjusted with a mineral acid such as citric acid, acetic acid or phosphoric acid.

Through extensive experiments and data screening, the inventor finds that when the concentration of histidine in the preparation of the invention is lower than 10mM, the buffering capacity of a buffering system is obviously limited, and when the concentration of histidine is higher than 30mM, the stability of the preparation is not improved.

One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's pharmaceutical 1Sciences 1uth edition, osol, A.Ed. (1980), may be included in the formulations of the invention so long as they do not adversely affect the desired characteristics of the formulation. 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; integrins such as EDTA; metal complexes (e.g., zn-protein complexes); biodegradable polymers such as polyvinyl acetate; and/or salt-forming counterions.

The formulations of the present invention may be prepared by combining the various components at certain concentrations by methods well known in the art.

The features mentioned above with reference to the invention, or the features mentioned with reference to the embodiments, can be combined arbitrarily. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.

The detection methods used in the following examples are illustrated below:

SEC purity, detection method of the Polymer:

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 e2695 2489 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 to 5.0mg/ml with mobile phase, centrifuging at 13000rpm for 10min, taking supernatant, transferring to a sample bottle, and placing in HPLC sample tray. Chromatographic conditions are as follows: the column temperature is 25 +/-2 ℃; the sample temperature is 10 +/-2 ℃; detecting the wavelength UV 280nm; the injection volume is 20 mu L; the flow rate was 0.5ml/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 degassing with ultrasound. And (3) mobile phase B:20mM phosphate buffer +200mM sodium chloride, pH 6.5. + -. 0.05. Filtering with 0.22 μm filter membrane, and degassing with ultrasound. And (3) chromatographic column: propac WCX-10, 4X 250mm, thermo Dionex 054993. A 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 temperature of the sample is 10 +/-2 ℃; the detection wavelength is UV 214nm; the injection volume is 20 mu L; the flow rate was 1.0ml/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. IEC purity results are reported as peak area percentages of the main peak.

A detailed description of conventional methods or methods conventional in the art is not included in the following examples. 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 the 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 type 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 a human IgG4 heavy chain and a kappa light chain to form the antibody, and 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

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

The histidine buffer system in the following examples refers to 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 were all commercially available unless otherwise specified.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 Effect of pH on formulations

5 kinds of buffer solutions with different pH values in the table 1 are prepared, and the following solution is obtained after the protein and the auxiliary materials are prepared. 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 1 pH 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 a 0.22 mu m filter membrane, subpackaging 1 ml/bottle into 2ml penicillin bottles, plugging, rolling a cover to obtain candidate prescription samples, placing the candidate prescription samples into 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

The results are shown in Table 4, the stability of each prescription is examined under the condition of 40 +/-2 ℃, and the change trends of the SEC purity and the IEC purity both 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 a 0.22 mu m filter membrane, subpackaging 1 ml/bottle into 2ml penicillin bottles, plugging, rolling a cover to obtain candidate prescription samples, placing the candidate prescription samples into a stability test box at 40 +/-2 ℃, sampling and inspecting at 1 week, 2 weeks, 4 weeks and 8 weeks, and inspecting indexes of SEC purity, IEC purity and insoluble particles.

TABLE 5 protein protectant and surfactant species screening prescription information

The results are shown in table 6, and the stability, insoluble particle and SEC purity of each prescription are examined under the condition of 40 ± 2 ℃, and the results further show that the stability can be effectively improved by adding the saccharide protective agent and polysorbate in the prescription; the variation trend of other detection items has no obvious difference among all the directions. In addition, comparing the results of the examination of 3-2 and 3-3, the addition of trehalose was slightly superior to the addition of sucrose. 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 adjuvants on the formulation

Preparing each formula solution in the table 7, respectively adding the protein into corresponding auxiliary materials, and then adjusting the protein concentration to 25mg/ml by using the auxiliary material solution prepared additionally 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 0.1mg/ml.

Table 8 examination of the amount of auxiliary materials

Example 5 accelerated stability test

An anti-PD-1 monoclonal antibody liquid formulation was prepared according to the following formulation: 25mg/ml of anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer, 95mg/ml of trehalose, 0.1mg/ml of polysorbate 80, and pH5.5 +/-0.5. 3 batches of the medicine are prepared, placed at 25 +/-2 ℃ for 6 months, sampled and detected for SEC purity and IEC purity at 0, 1, 2, 3 and 6 months, and the results are shown in table 9, and the results show that the medicine purity meets the standard requirements after the acceleration for 6 months.

TABLE 9 accelerated stability results

The data in Table 9 show that the SEC purity decreases from 99.33% to only 98.83% with a decrease of only about 0.5% when left at 25. + -. 2 ℃ for 6 months; the IEC purity decreased from 46.83% to 44.17% with a variation of about 5%, indicating that the formulations of the invention have excellent stability, i.e. can be stored at 25 ℃ for at least 6 months.

Example 6 Long term stability Studies

An anti-PD-1 monoclonal antibody liquid preparation is prepared according to the following preparation formula: 25mg/ml of anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer, 95mg/ml of trehalose, 0.1mg/ml of polysorbate 80, and 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

The data in Table 10 show that the SEC purity decreases from 99.33% to only 98.83% with a decrease of only about 0.5% when left at 5 + -3 deg.C for 24 months; the IEC purity increased from 46.83% to 48.63% with a variation of about 3.8%, not only with no decrease but with a slight increase, indicating that the formulations of the invention have excellent stability, i.e. can be stored at 25 ℃ for at least 6 months.

Further analysis of the results as shown in fig. 1, it can be seen from fig. 1 that the purity SEC of the anti-PD-1 monoclonal antibody preparation of the present invention has a predicted expiration date of 154 months, IEC purity does not decrease during long-term stabilization, and insoluble particle data does not significantly change, thus supporting an expiration date of 60 months (expiration date, i.e., the period under long-term stabilization conditions).

Discussion:

stability of the antibody formulation is of critical importance. Several prior studies have shown that different formulations lead to differences in the stability of the formulation for different antibody products.

After a large number of screens, the optimized preparation has excellent stability:

(a) The accelerated stability data shown in table 9 show that the change in the main peak of the charge isomer (IEC purity) is reduced by about 2% (from 46.83% to 44.17%), which is significantly better than the currently reported analogous formulations.

(b) The long term stability results shown in table 10 show that there is no downward trend in the change in charge Isomers (IEC). The antibody preparation formula of the invention is shown to be superior to the similar preparation reported at present.

(c) The antibody formulations of the present invention do not contain antioxidants (e.g., methionine), but still have excellent long term stability.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the appended claims of the present application.

Claims

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 a 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 or a histidine-histidine hydrochloride buffer solution;

the protein protective agent is selected from trehalose, sucrose or mannitol;

the surfactant is selected from polysorbate 20 or polysorbate 80;

the pH of the liquid preparation is 5.0-7.0.
The liquid formulation of claim 1, wherein the anti-PD-1 monoclonal antibody is present at a concentration of 25mg/ml.
The liquid formulation of claim 1, wherein the buffer is histidine-histidine hydrochloride buffer or acetate buffer, and the buffer concentration is 10mM.
The liquid formulation of claim 1, wherein the protein protectant is trehalose, sucrose, or mannitol, and the concentration of the protein protectant is 40-95mg/ml;

preferably, the protein protective agent is trehalose or sucrose with a concentration of 80mg/ml to 95mg/ml, or mannitol with a concentration of 40mg/ml.
The liquid formulation of claim 1, wherein the surfactant is polysorbate 20 or polysorbate 80, and wherein the surfactant concentration is 0.1mg/ml.
The liquid formulation of claim 1, wherein the liquid formulation has a pH of 5.0 to 6.0.
The liquid formulation of claim 1, wherein the human PD-1 monoclonal antibody is an IgG 4-type antibody.
The liquid formulation of claim 1, wherein the formulation comprises:

anti-PD-1 monoclonal antibody with the concentration of 20-40mg/ml, buffer solution with the concentration of 10-30mM, protein protective agent with the concentration of 40-100mg/ml and surfactant with the concentration of 0.1 mg/ml;

the buffer solution is histidine-histidine hydrochloride buffer solution;

the protein protective agent is selected from trehalose, sucrose or mannitol;

the surfactant is polysorbate 80;

the pH of the liquid preparation is 5.0-6.0.
The formulation of any one of claims 1 to 8, wherein the formulation has one or more characteristics selected from the group consisting of:

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

(b) The liquid formulation is stored at 25 ℃ for at least 6 months.
The formulation of claim 9, wherein the formulation has one or more characteristics selected from the group consisting of:

(a) The liquid preparation is stored for 6 months at 25 +/-2 ℃, and the SEC purity is more than or equal to 97 percent, preferably more than or equal to 98 percent, and more preferably more than or equal to 98.5 percent; and/or IEC purity is not less than 40%, preferably not less than 42%, more preferably not less than 44%;

(b) The liquid preparation is stored for 24 months at 5 +/-3 ℃, and the SEC purity is more than or equal to 97 percent, preferably more than or equal to 98 percent, and more preferably more than or equal to 98.5 percent; and/or the IEC purity is not less than 40%, preferably not less than 42%, more preferably not less than 44% or not less than 46%.
The liquid formulation of claim 9 or 10, wherein the liquid formulation is for administration by injection.
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.
The use of claim 12, wherein the disease associated with the PD-1 signaling pathway comprises cancer or an autoimmune disease.
A kit, comprising:

the antibody liquid preparation of any one of claims 1 to 8; and

a container for holding the liquid formulation.
The kit of claim 14, wherein the kit further comprises instructions for use.