Liquid preparation of anti-RANKL antibody

Abstract

The invention provides a liquid preparation of an anti-RANKL antibody. The liquid preparation comprises the following components: 60-80 g / l of the anti-RANKL antibody, 50-100 g / l of sugar or sugar alcohol, 0.1-0.3 g / l of a surfactant and 10-30 mM / l of amino acid, the buffer system is selected from a histidine-histidine hydrochloride buffer system. The aggregation and degradation of protein can be effectively reduced, and the stability is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a liquid preparation of anti-RANKL antibody. BACKGROUND

[0002] RANKL (Receptor Activator of NF-κB Ligand) is a member of the tumor necrosis factor superfamily. RANKL (TNFSF11) is a type II transmembrane protein and is a ligand for the tumor necrosis factor NF-κB (RANK) receptor. RANKL is an activator of NF-κB. RANKL can bind to RANK and induce differentiation of monocyte / macrophage lineage cells into osteoclasts, further leading to maturation of osteoclast precursors and inhibition of osteoclast apoptosis. Osteoclasts have bone-eroding effects and can destroy bone and be absorbed, which is an important part of bone turnover. Therefore, anti-RANKL antibodies can be used for the treatment of osteoporosis.

[0003] In addition, studies have shown that the RANKL / RANK pathway can promote bone metastasis by activating osteoclasts, and by inhibiting RANKL, bone resorption and tumor growth caused by tumor metastasis can be prevented, and anti-RANKL antibodies also have application value in tumor treatment.

[0004] Compared with traditional organic and inorganic drugs, anti-RANKL antibodies are protein macromolecules, which are relatively larger in size and more complex in structure. Protein drugs are generally administered in the form of injections, and the drug molecules need to be dissolved in a certain solution. Proteins in aqueous solution can have chemical or physical instability, leading to changes in the surface charge, structure and function of the antibody, which can affect the safety and effectiveness of the drug. Currently, marketed antibody formulation systems generally contain three types of substances: buffers, sugar or sugar alcohol stabilizers, and surfactants. However, over time, antibodies still have problems such as decreased stability, degradation, and increased aggregates. SUMMARY

[0005] In order to maintain the stability, safety and biological activity of anti-RANKL antibodies, the present application provides a liquid preparation of anti-RANKL antibodies, which optimizes the preparation by screening and is a stable and effective formulation of anti-RANKL antibodies.

[0006] In order to achieve the purpose of the present application, the technical scheme adopted by the present application is: A liquid preparation of anti-RANKL antibody, comprising the following components: Anti-RANKL antibody 60-80 g / l Sugar or sugar alcohol 50-100 g / l Surfactant 0.1-0.3 g / l Amino acid 10-30 mM / l.

[0007] The buffer system is selected from the group consisting of histidine-histidine hydrochloride.

[0008] In the development of the formulation, the kind of buffer system and pH value have great influence on the stability of the formulation, so it is important to select a suitable buffer system and maintain a specific pH range for the anti-RANKL antibody formulation. The antibody is easy to precipitate near the isoelectric point, so it is necessary to select a suitable pH range of the buffer system to maintain the physicochemical stability of the protein.

[0009] The pH value of the buffer system of the present application is 5.5-6.5.

[0010] The sugar or sugar alcohol of the present application is selected from any one of sucrose, trehalose, sorbitol and mannitol, preferably trehalose, and the content is 80 g / l.

[0011] The amino acid of the present application is selected from any one of glycine, proline, arginine and methionine, preferably methionine, and the content is 10-20 mM / l.

[0012] The sugar and polyol in the formulation of the present application act as stabilizers, have a preferential exclusion effect, and can stabilize the native conformation of the protein, thereby improving the stability of the formulation.

[0013] Amino acids can form multiple hydrogen bonds with proteins as hydrogen bond donors or acceptors to stabilize them. In addition, charged amino acids can have electrostatic interactions with oppositely charged side chains. Such interactions can result in an increase in protein solubility and / or conformational stability, while minimizing protein-protein interactions.

[0014] The present application has been screened, and it has been found that methionine can effectively reduce the increase in aggregates after being placed at 40℃ for 4 weeks, so methionine is selected as the first stabilizer. Trehalose can effectively reduce the increase in aggregates after being placed at 40℃ for 4 weeks, so trehalose is selected as the second stabilizer.

[0015] The surfactant of the present application is selected from polysorbate 20, polysorbate 80 and poloxamer 188, preferably polysorbate 80.

[0016] Surfactants can competitively accumulate on surfaces or interfaces and / or directly interact with proteins, thereby minimizing the interaction of proteins with surfaces or interfaces and with each other. Polysorbate 80 as a surfactant can minimize the adsorption of proteins on the air / water interface, thereby minimizing aggregation.

[0017] The present application optimizes the anti-RANKL antibody formulation through screening, and it has been found that trehalose and methionine can reduce protein aggregation and degradation, effectively protect the stability of the active ingredient of the protein in the formulation, and make the liquid formulation have good stability. DETAILED DESCRIPTION

[0018] In order to more clearly and specifically illustrate the technical solutions of the present application, the present application is further described below through relevant examples. The following examples are merely used to specifically illustrate the implementation methods of the present application, and do not limit the protection scope of the present application. Examples

[0019] The present application carries out screening experiments of amino acids, and the pH of the prepared liquid preparation is 6.0, which is composed of the following components: 60 g / L denosumab as an anti-RANKL human monoclonal antibody as a protein drug, polysorbate 80 (a surfactant), a buffer, and a stabilizer (an amino acid).

[0020] Preparation of anti-RANKL antibody preparation: The purified anti-RANKL antibody is subjected to ultrafiltration and replacement into a histidine-histidine hydrochloride buffer solution, and is further concentrated. The auxiliary material mother liquor of trehalose, methionine, and polysorbate 80 is added according to the prescription concentration. Finally, the protein concentration is adjusted to the prescription concentration by using the histidine-histidine hydrochloride buffer solution.

[0021] Table 1: Prescription of liquid preparation in each group

[0022] In order to determine the stability of the anti-RANKL antibody in the liquid preparation of various combinations, the liquid preparation is stored at 40℃ for 1 month, and the amount of high molecular polymer (%) and the amount of free monomer (%) in the liquid preparation are measured by SEC (size exclusion chromatography) and NR-cEG (non-reduced capillary gel electrophoresis), respectively.

[0023] Table 2: SEC detection results of amino acid screening study

[0024] Table 3: NR-cGE detection results of amino acid screening study

[0025] As can be seen from Tables 2 and 3, methionine as an amino acid stabilizer has the highest free monomer % and the lowest polymer % after the liquid preparation is placed for 1 month, indicating that the addition of methionine can more improve the stability of the liquid preparation than other amino acids, and the stability is the highest when the addition amount of methionine is 10-20 mM.

[0026] The present application further carries out screening experiments of sugars or sugar alcohols, and the prescription of the liquid preparation in each group is shown in Table 4.

[0027] Table 4: Prescription of liquid preparation in each group

[0028] Table 5. SEC detection results of sugar or sugar alcohol screening study

[0029] Table 6. NR-cGE detection results of sugar or sugar alcohol screening study

[0030] From Table 5 and Table 6, it can be seen that when methionine is used as the first stabilizer and trehalose is used as the second stabilizer, the aggregate content of the prepared liquid preparation is 0, and the stability after 1 month is higher than that of group 12-14, especially when the content of trehalose is 80 g / l, the aggregate content after 1 month is only 0.48%.

[0031] Example 2 A liquid preparation of an anti-RANKL antibody comprises the following components: Anti-RANKL antibody 60 g / l Trehalose 80 g / l Polysorbate 80 0.1 g / l Methionine 10 mM / l.

[0032] The buffer system is a histidine-histidine hydrochloride buffer system, and the pH value of the buffer system is 5.5.

[0033] Example 3 A liquid preparation of an anti-RANKL antibody comprises the following components: Anti-RANKL antibody 60 g / l Trehalose 80 g / l Polysorbate 80 0.3 g / l Methionine 20 mM / l.

[0034] The buffer system is a histidine-histidine hydrochloride buffer system, and the pH value of the buffer system is 6.5.

[0035] Example 4 A liquid preparation of an anti-RANKL antibody comprises the following components: Anti-RANKL antibody 60 g / l Trehalose 100 g / l Polysorbate 80 0.2 g / l Methionine 20 mM / l.

[0036] The buffer system is a histidine-histidine hydrochloride buffer system, and the pH value of the buffer system is 6.0.

[0037] Example 5 A liquid preparation of an anti-RANKL antibody comprising the following components: Anti-RANKL antibody 80 g / l Trehalose 80 g / l Polysorbate 80 0.2 g / l Methionine 15 mM / l.

[0038] The buffer system is a histidine-histidine hydrochloride buffer system, and the pH value of the buffer system is 6.0.

[0039] Example 6 A liquid preparation of an anti-RANKL antibody comprising the following components: Anti-RANKL antibody 70 g / l Trehalose 80 g / l Polysorbate 80 0.2 g / l Methionine 20 mM / l.

[0040] The buffer system is a histidine-histidine hydrochloride buffer system, and the pH value of the buffer system is 6.0.

[0041] Table 7 SEC detection results of the liquid preparation of the example

[0042] The above-described examples only express the specific embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A liquid formulation of an anti-RANKL antibody, characterized in that, Includes the following components: Anti-RANKL antibody 60-80g / l Sugar or sugar alcohol 50-100g / l Surfactant 0.1-0.3 g / L Amino acids 10-30 mM / l; The buffer system is selected from the histidine-histidine hydrochloride buffer system.

2. The liquid formulation of the anti-RANKL antibody according to claim 1, characterized in that: The pH value of the buffer system is 5.5 to 6.

5.

3. The liquid formulation of the anti-RANKL antibody according to claim 1, characterized in that: The sugar or sugar alcohol is selected from any one of sucrose, trehalose, sorbitol and mannitol.

4. The liquid formulation of the anti-RANKL antibody according to claim 3, characterized in that: The sugar or sugar alcohol is trehalose, with a content of 80 g / L.

5. The liquid formulation of the anti-RANKL antibody according to claim 1, characterized in that: The amino acid is selected from any one of glycine, proline, arginine, and methionine.

6. The liquid formulation of the anti-RANKL antibody according to claim 5, characterized in that: The amino acid in question is methionine, with a content of 10-20 mM / l.

7. The liquid formulation of the anti-RANKL antibody according to claim 1, characterized in that: The surfactant is selected from any one of polysorbate 20, polysorbate 80, and poloxamer 188.

8. The liquid formulation of the anti-RANKL antibody according to claim 7, characterized in that: The surfactant is polysorbate 80.

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