CN114099672A

CN114099672A - Pharmaceutical composition of anti-RANKL humanized monoclonal antibody with enhanced stability

Info

Publication number: CN114099672A
Application number: CN202210103689.5A
Authority: CN
Inventors: 林军; 张莉娜; 袁梵雨; 贺晋艳
Original assignee: GENOR BIOPHARMA CO Ltd
Current assignee: GENOR BIOPHARMA CO Ltd
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-03-01
Anticipated expiration: 2042-01-28
Also published as: CN114099672B

Abstract

The present disclosure relates to a pharmaceutical composition of an anti-RANKL humanized monoclonal antibody with enhanced stability. Specifically, the pharmaceutical composition of the present disclosure comprises an anti-RANKL humanized monoclonal antibody, a buffer, a protective agent and/or an osmolality adjusting agent, and a surfactant. The pharmaceutical compositions of the present disclosure have enhanced stability under various conditions of routine storage, high temperature, shaking, freeze-thawing, transportation, and the like.

Description

Pharmaceutical composition of anti-RANKL humanized monoclonal antibody with enhanced stability

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a pharmaceutical composition of an anti-RANKL humanized monoclonal antibody with enhanced stability.

Background

The Nuclear Factor-kappa B Receptor Activator Ligand (Receptor Activator of Nuclear Factor kappa-B Ligand, "RANKL") is a member of the tumor necrosis Factor superfamily, which binds to the Nuclear Factor-kappa B Receptor Activator expressed on the surface of osteoclasts ("RANK") to promote differentiation and maturation of osteoclasts. RANKL is a homotrimeric membrane-associated protein typical of osteoblasts and activated T cells, and can be secreted by activated T cells and the like by proteolytic cleavage or alternative splicing, with high expression in lymph node, thymus, breast and lung tissues, and low expression in spleen and bone marrow and other tissues. Most factors known to stimulate osteoclast formation and activation induce the expression of RANKL by myeloid stromal cells. Synoviocytes and activated T cells in arthritic joints express RANKL and, together with Interleukins (IL) -1 and Tumor Necrosis Factor (TNF), promote fusion of osteoclast precursors to form osteoclasts, causing some degree of joint destruction. RANKL can also stimulate osteoclast precursors to enter the blood circulation. Under RANKL induction, osteoclast activation links bone remodeling with regulation of hematopoiesis.

Blocking RANKL/RANK signaling inhibits osteoclast formation and activity, blocks bone resorption, and thus treats osteoporosis, which has proven to be a viable route. Denosumab, a human-derived anti-human RANKL monoclonal antibody obtained by immunizing human IgG (immunoglobulin G) transgenic mouse XenoMouse, produced by Amgen, usa, is approved by the FDA for the treatment of postmenopausal osteoporosis in women, and EMEA for the treatment of bone loss associated with prostate cancer. The clinical effectiveness of Denosumab antibodies led to the final confirmation of the anti-RANKL monoclonal antibody as a target for drugs in the treatment of a range of bone metabolic diseases with increased osteoclast activity leading to bone loss.

Compared with the traditional synthesized small molecule drugs, the monoclonal antibody is a macromolecular substance and has a complex structure. The physicochemical properties of a monoclonal antibody depend not only on the composition of the amino acids in its primary structure, but also on the higher structure formed by folding it in a specific manner in a solvent (usually water), which structure also determines the pharmacodynamic properties of the pharmaceutical protein. Maintaining the high-order structure of an antibody is an essential requirement for exerting its biological activity. The stability of the higher order structure of a protein depends on the protein itself and on the interaction of the protein with its solvent and other solutes in the solvent. For monoclonal antibodies, finding a formulation that facilitates the stabilization of the higher order structure of its protein and facilitates drug administration is critical to biopharmaceutical formulations. Furthermore, improving the stability of biopharmaceutical formulations while maintaining high protein concentrations is particularly challenging.

Disclosure of Invention

In order to solve the above problems, the present disclosure provides a novel pharmaceutical composition of an anti-RANKL humanized monoclonal antibody (anti-RANKL monoclonal antibody for short) with enhanced stability, which makes the antibody formulation more stable and longer in storage time.

In one aspect, the present disclosure relates to a pharmaceutical composition comprising:

(a) anti-RANKL humanized monoclonal antibodies;

(b) a buffering agent;

(c) a protectant and/or osmoregulator;

(d) a surfactant;

wherein the buffer is citric acid/sodium citrate, glutamic acid/sodium glutamate, acetic acid/sodium acetate or histidine/histidine hydrochloride, preferably histidine/histidine hydrochloride; the protective agent and/or the osmotic pressure regulator is one or more of trehalose, sucrose and proline, and is preferably used in combination with sucrose and proline; the surfactant is polysorbate 80 (Tween 80) or polysorbate 20 (Tween 20), preferably polysorbate 80.

In a preferred embodiment, the anti-RANKL humanized monoclonal antibody is a full length IgG2 antibody that binds to a RANKL antigen comprising a light chain variable region sequence selected from the group consisting of SEQ ID. number 1 and a heavy chain variable region sequence of SEQ ID. number 2.

SEQ ID. No. 1：

NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQRPGQAPRLLIYLASNLESGVPARFSGSGSRTDFTLTISSVEAEDEATYYCQQDNEDPYTFGGGTKLEIK

SEQ ID.No.2：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWLQWVRQAPGQGLEWMGAIYPGPGNTKYTQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARRGSRRGIAYWGQGTLVTVSA

In a preferred embodiment, the anti-RANKL humanized monoclonal antibody has the full length of the light chain as shown in SEQ id No.3 and the full length of the heavy chain as shown in SEQ id No. 4.

SEQ ID.No.3：

NIVLTQSPASLSVSLGQRATISCRASESVDSYGRSFMHWYQQRPGQAPRLLIYLASNLESGVPARFSGSGSRTDFTLTISSVEAEDEATYYCQQDNEDPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID.No.4：

QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWLQWVRQAPGQGLEWMGAIYPGPGNTKYTQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARRGSRRGIAYWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In a preferred embodiment, the anti-RANKL humanized monoclonal antibody has a protein concentration of 60-100 g/L, such as 60-80 g/L, more specifically, such as 70 g/L.

In a preferred embodiment, the concentration of the protectant and/or osmolyte is from 10 to 90 g/L, such as from 50 to 70 g/L.

In a preferred embodiment, the surfactant is present at a concentration of 0.01 to 0.15g/L, such as 0.05 to 0.1 g/L.

In a preferred embodiment, the buffer is present at a concentration of 1 to 5g/L, such as 3 to 4 g/L.

In a preferred embodiment, the pH of the pharmaceutical composition is from 4.5 to 7.5, preferably from 5.0 to 6.0, more preferably about 5.8.

In certain embodiments, the pharmaceutical composition comprises:

-60-80 g/L of an anti-RANKL humanized monoclonal antibody;

-1.0-1.2 g/L L-histidine;

-2.5-3.0 g/L of L-histidine monohydrochloride monohydrate

35.0 to 45.0 g/L of sucrose;

-10.0-15.0 g/L proline;

-0.08-0.12 g/L polysorbate 80.

In a preferred embodiment, the pharmaceutical composition is an aqueous solution having a pH of 5.8 of:

in another aspect, the disclosure relates to the use of a pharmaceutical composition in the manufacture of a medicament for the treatment of a bone metabolic disease resulting in bone loss due to enhanced osteoclast activity.

In a preferred embodiment, the bone metabolic disease is osteoporosis.

THE ADVANTAGES OF THE PRESENT INVENTION

The pharmaceutical composition of the present disclosure has at least the following advantages:

the pharmaceutical composition disclosed by the invention has enhanced stability under various conditions of conventional preservation, high temperature, oscillation, freeze thawing, transportation and the like, is beneficial to storage of the medicine, and can prolong the storage period of the medicine;

the pharmaceutical composition of the present disclosure contains a high concentration of anti-RANKL mab.

Detailed Description

Definition of

To facilitate an understanding of the invention, certain terms of the invention are defined or described.

Unless otherwise indicated or defined, all terms used have the ordinary meaning in the art that will be understood by those skilled in the art.

SEC-HPLC: size exclusion high performance liquid chromatography

HMW: high molecular weight peak group (mer)

LMW: low molecular weight peak group (fragment)

CEX-HPLC: cation exchange high performance liquid chromatography

And (3) acid: set of acidic peaks

Basic: set of basic peaks

Main: main peak group

rCE-SDS: reduced capillary gel electrophoresis method

HC: heavy chain

LC: light chain

NGHC: sugar-free heavy chain

XMW: impurities or degradation fragments

FlowCAM: combining flow cytometry into a digital imaging microscope, and performing particle analysis based on flow dynamic imaging of image analysis method

NT: not detected

SD: standard deviation of

UV 280: absorbance at 280nm of UV spectrophotometer for quantitation of protein content (UV absorption coefficient method)

The term "stability" means that the antibody in a pharmaceutical formulation or pharmaceutical composition substantially retains its physical and/or chemical and/or biological stability upon storage.

The term "buffer" refers to a substance that maintains the pH of a solution substantially constant over a range. As used herein, the buffering agent includes, but is not limited to, citric acid, sodium citrate, glutamic acid, sodium glutamate, acetic acid, sodium acetate, histidine hydrochloride, phosphate buffered saline, Tris buffer, and the like.

As used herein, the term "protectant and/or osmo-regulator" includes, but is not limited to, mannitol, trehalose, sucrose, proline, and the like, preferably a combination of sucrose and proline.

As used herein, the term "surfactant" includes, but is not limited to, polysorbate 80 (tween 80) or polysorbate 20 (tween 20) and the like, preferably polysorbate 80.

The term "bone disease" includes osteoporosis, osteoarticular bone destruction caused by rheumatoid arthritis, bone destruction caused by tumor bone metastasis, bone destruction caused by megakaryocytoma growth, and other pathological changes such as bone loss or destruction due to RANKL-induced osteoclastogenesis. Bone diseases as referred to herein primarily refer to bone loss diseases or bone metabolism diseases. Exemplary bone metabolic diseases include, but are not limited to, osteoporosis, osteomalacia, and the like.

The term "osteoporosis" is a complex metabolic disease of bone, whose development is mainly caused by an imbalance in bone formation and bone resorption, and is characterized by low bone density and defects in bone tissue microstructure, resulting in increased bone fragility and susceptibility to fracture. Osteoporosis includes, but is not limited to, primary osteoporosis, endocrine osteoporosis (including, but not limited to, hyperthyroidism, hyperparathyroidism, cushing's syndrome, and acromegaly), genetic and congenital forms of osteoporosis (including, but not limited to, osteogenesis imperfecta, homocystinuria, menkes' syndrome, lysine-wearing syndrome), and osteoporosis due to acromegaly immobilization; adult and juvenile Paget's disease (osteitis deformans); osteomyelitis, a infectious lesion of bone that results in bone loss; hypercalcemia, including but not limited to hypercalcemia resulting from solid tumors (including but not limited to breast, lung and kidney) and hematological malignancies (including but not limited to multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, and hypercalcemia associated with thyroid function nonadjustment and renal function disorders; osteopenia, including but not limited to osteopenia after surgery, osteopenia induced by administration of a drug, osteopenia associated with small and large bowel disease, and osteopenia associated with chronic liver and kidney disease; osteonecrosis, i.e., osteocyte death, including but not limited to osteonecrosis associated with traumatic injury, osteonecrosis associated with gaucher's disease, osteonecrosis associated with sickle cell anemia, osteonecrosis associated with systemic lupus erythematosus, osteonecrosis associated with rheumatoid arthritis, osteonecrosis associated with periodontal disease, osteonecrosis associated with osteolytic metastasis, osteonecrosis associated with other conditions; and cartilage loss and joint erosion associated with rheumatoid arthritis.

Examples

For a better understanding of the present invention, the present invention will be described in detail with reference to the following examples, but it should be understood that these examples are only illustrative of the present invention and are not intended to limit the present invention.

In the present invention, all operations are carried out at room temperature and normal pressure unless otherwise specified.

The anti-RANKL humanized monoclonal antibody used in the embodiment of the disclosure is an anti-RANKL humanized monoclonal antibody independently developed by the applicant; it is a full length IgG2 antibody, which binds to RANKL antigen and comprises a light chain variable region sequence selected from SEQ ID. number 1 and a heavy chain variable region sequence of SEQ ID. number 2; the total length of the light chain is shown as SEQ ID No. 3; the whole length of the heavy chain is shown as SEQ ID No. 4. Sucrose was purchased from J.T.Baker, USA and Pfansteehl, proline was purchased from Shanghai cooperative and amino acids, Inc., L-histidine hydrochloride monohydrate, polysorbate 80 was purchased from J.T.Baker, USA.

Exemplary pharmaceutical compositions of the present disclosure (referred to in the following examples as "anti-RANKL mab pharmaceutical compositions") are composed as follows:

components	Content (g/L)
		anti-RANKL monoclonal antibody	70.0
L-histidine	1.13
		Monohydrate L-histidine hydrochloride	2.66
Sucrose	40.0
		ProlineAcid(s)	13.9
Polysorbate 80	0.1
		pH	5.8

Example 1 conventional stability assay

The anti-RANKL monoclonal antibody drug composition is subjected to a room temperature oscillation 72-hour experiment, a freezing and thawing 5-cycle experiment at room temperature of-20 ℃/4 ℃, a long-term stability experiment at 4 ℃ and an accelerated stability experiment at 40 ℃. Wherein the oscillation experiment conditions are as follows: sampling after standing at 25 ℃ and 180 rpm for 72 hours; the conditions of the freeze-thaw experiment are as follows: samples were taken after 5 cycles of-20 ℃/room temperature freeze-thaw. Preliminary analytical tests were performed by SEC-HPLC, CEX-HPLC, rCE-SDS, FlowCAW, using biological activity as a standard for the final determination of stability. The results are shown in Table 1.

Table 1: results of the respective test items for routine stability

As can be seen from table 1, no significant change in particle or molecular size heterogeneity was observed in the anti-RANKL mab pharmaceutical compositions whether stored at 4 ℃ in the dark for 3 months or subjected to shaking or freeze-thawing; no change in biological activity results was observed in the case of storage at 40 ℃ for 1 or 2 months in the dark; furthermore, 5 cycles of-20 ℃/room temperature freeze-thaw with room temperature shaking for 72 hours, no significant change was observed in the molecular size heterogeneity and insoluble microparticles (data not shown).

The results show that the anti-RANKL monoclonal antibody medicinal composition has excellent physicochemical stability and is suitable for being used as a clinical preparation.

Example 2 transport stabilityInvestigation of

In the embodiment, a scientific basis is provided for the packaging, storage, transportation conditions and the like of the medicine by investigating the rule of the quality change of the anti-RANKL monoclonal antibody medicine composition under the influence of the transportation conditions.

Table 2: transportation stability test results

The above results indicate that after transportation and storage for 6 months in a forward or reverse manner, respectively, the anti-RANKL mab pharmaceutical composition of the present disclosure has no significant change in both molecular size and charge heterogeneity, has excellent stability, and no significant decrease in biological activity is observed.

Example 3 injection stability Studies

In the medicinal protein injection, when the liquid medicine passes through the needle from the syringe, the water pressure in the needle pipeline is greatly increased due to the sharp reduction of the diameter of the needle, which is not favorable for the colloidal stability of the protein. The anti-RANKL monoclonal antibody drug composition is repeatedly sucked by a syringe, and the physicochemical properties of the sucked liquid medicine are compared.

The anti-RANKL mab pharmaceutical composition was aspirated 10 times with a 1 mL syringe and stored at 4 ℃ for 3 hours and 24 hours, respectively. The results are shown in Table 3.

Table 3: experimental results on injection stability

As can be seen from table 3, after 10 times of pumping, the anti-RANKL mab pharmaceutical composition sample of the present disclosure showed no significant change in protein concentration and SEC-HPLC purity, insoluble particles were also at a low level, and no destabilization of the protein due to shear force was observed.

The above examples show that the anti-RANKL mab pharmaceutical composition of the present disclosure achieves unexpected effects, and has enhanced stability under various conditions of conventional storage, high temperature, shaking, freeze-thawing, transportation, and the like. In addition, the pharmaceutical composition disclosed by the invention contains the anti-RANKL monoclonal antibody with high concentration (such as 70.0 g/L), and has the remarkable advantage of high concentration of the active ingredient. The pharmaceutical composition disclosed by the invention realizes high-concentration active ingredients, has excellent stability and has important clinical application value.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Various changes and modifications may be made to the invention without departing from the spirit and scope of the invention, and such changes and modifications are intended to be within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Claims

1. A pharmaceutical composition comprising:

(a) anti-RANKL humanized monoclonal antibodies;

(b) a buffering agent;

(c) a protectant and/or osmoregulator;

(d) a surfactant;

wherein the buffer is selected from histidine/histidine hydrochloride, citric acid/sodium citrate, glutamic acid/sodium glutamate, acetic acid/sodium acetate;

wherein the protective agent and/or the osmotic pressure regulator is one or more of trehalose, sucrose and proline;

wherein the surfactant is polysorbate 80 or polysorbate 20.

2. The pharmaceutical composition according to claim 1, wherein the anti-RANKL humanized monoclonal antibody comprises a light chain variable region sequence selected from the group consisting of SEQ ID. number 1 and a heavy chain variable region sequence of SEQ ID. number 2.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-RANKL humanized monoclonal antibody has a protein concentration of 60-100 g/L.

4. The pharmaceutical composition of claim 1 or 2, wherein the buffer is histidine/histidine hydrochloride.

5. The pharmaceutical composition according to claim 4, wherein the buffer is present in an amount of 1-5 g/L, preferably 3-4 g/L.

6. The pharmaceutical composition according to claim 1 or 2, wherein the protective agent and/or tonicity modifier is a combination of proline and sucrose.

7. The pharmaceutical composition according to claim 6, wherein the concentration of the protective agent and/or the osmolality adjusting agent is 10-90 g/L, preferably 50-70 g/L.

8. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the surfactant is 0.01-0.15 g/L, preferably 0.05-0.1 g/L.

9. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is 4.5 to 7.5, preferably 5.0 to 6.0.

10. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises:

-60-80 g/L of an anti-RANKL humanized monoclonal antibody;

-1.0-1.2 g/L L-histidine;

-2.5-3.0 g/L of L-histidine monohydrochloride monohydrate

35.0 to 45.0 g/L of sucrose;

-10.0-15.0 g/L proline;

-0.08-0.12 g/L polysorbate 80.

11. The pharmaceutical composition according to claim 1 or 2, which is an aqueous solution having a pH of 5.8 of the following components:

components Content (g/L) anti-RANKL humanized sheetCloning antibodies 70.0 L-histidine 1.13 Monohydrate L-histidine hydrochloride 2.66 Sucrose 40.0 Proline 13.9 Polysorbate 80 0.1

。

12. Use of a composition according to any one of claims 1 to 11 in the manufacture of a medicament for the treatment of bone disease.

13. The use according to claim 12, wherein the bone disease is a metabolic disease of bone resulting in bone loss due to enhanced osteoclast activity.

14. The use according to claim 13, wherein the bone metabolic disease is osteoporosis.