CN109745559A - The liquid preparation of the monoclonal antibody of anti-human IL-17A - Google Patents

Abstract

The invention belongs to antibody preparation fields, more specifically, the invention discloses a kind of liquid preparation of the monoclonal antibody of anti-human IL-17A and its applications.The liquid preparation of anti-human IL-17A monoclonal antibody provided by the invention includes anti-human IL-17A monoclonal antibody, surfactant, sugar and amino acid, it is stable during manufacture, storage and administration, with lower aggregation, and it is suitble to high concentration (such as subcutaneous administration) application.The liquid preparation of stable anti-human IL-17A monoclonal antibody of the invention can effectively apply in the preparation for treating the drug of immune-mediated inflammatory reaction.

Description

The liquid preparation of the monoclonal antibody of anti-human IL-17A

Technical field

The present invention relates to biomedicine fields, and in particular to a kind of liquid preparation of the monoclonal antibody of anti-human IL-17A.

Background technique

Interleukin-17 (Interleukin 17, IL-17) is a kind of inflammation cytokines, mainly by complementary T Cell 17 (T helper 17, Th17) secretion, other T cells and congenital immunity cell such as mast cell and neutrophil(e) granule are thin Born of the same parents can also secrete certain IL-17, play a significant role in a variety of inflammatory reactions and autoimmune disease pathologic process. IL-17 family member includes: IL-17A, IL-17F, IL-17B, IL-17C, IL-17D and heterologous two of homodimer form IL-17A/F, IL-17E/IL-25 of dimer form, in addition, there are two unnamed members.IL-17 receptor (IL- 17receptor, IL-17R) family includes: IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE, wherein homologous Dimer IL-17A and homodimer IL-17F and IL-17A/F heterodimer collective effect are in IL-17RA and IL-17RC. In vitro, T cells activate under the stimulation of antigen and costimulatory molecules, by transforming growth factor-β (transforming Growth factor- β, TGF-β), the cytokine inductions such as IL-6, IL-23 break up Th17, Th17 cell secrete IL-17A, IL-17F cell factor.IL-17 and IL-17R compound pass through signal transduction complex IL-17R-Act1- tumor necrosis factor Receptor associated factor 6 (TNF receptor associated factor 6, TRAF6) activates downstream NF- κ B, the end c-jun N- The signal paths such as kinases (c-jun N-terminal kinase, JNK) participate in inflammatory reaction.

Studies have shown that IL-17 takes part in every pathological reaction [Nat Rev Drug of autoimmune disease comprehensively Discov.2012.11 (10): 763-76]: IL-17 stimulating endothelial cell secretes IL-6 and IL-8, promotes thrombosis；Simultaneously Epithelial cell, fibroblast, macrophage and Dendritic Cells is induced to secrete inflammatory cytokine, induction inflammatory reaction hair It is raw；After it acts on cartilage cell, nitric oxide production expression is raised, cartilage destruction is caused；Induced osteogenesis cell secretes core simultaneously Factor κ-B ligand receptor activator (Receptor activator of nuclear factor kappa-B ligand, RANKL), promote osteoclastic effect, lead to bone injury.

Psoriasis also known as psoriasis are a kind of chronic inflammatory skins, and whole world morbidity is 1%-3%, are fallen ill in China Rate is 0.1%-0.3%, accounts for the 10% of global incidence.Currently, still lacking permanently effective treatment method.Studies have shown that IL- 17 accesses are that splendid psoriasis intervenes target spot, for IL-17A antibody after psoriasis patients were using 12 weeks, PASI75 (silver Consider to be worth doing sick skin lesion improvement reach 75%) response rate can reach 83.3% [The New England Journal of Medicine, 2014,371:326-338.].Market report shows that anti-IL-17A antibody is expected to become in 2018 year sale more than 1,000,000,000 beauty The heavy bomb drugs of gold.

In general, protein has very short half life, and (such as it is not suitable for temperature, water-gas being exposed to various factors Interface, high pressure, physical/mechanical stress, organic solvent and microbial contamination) undergo denaturation (such as to assemble, dissociate and be adsorbed on later Vessel surface).Therefore, albuminate loses inherent physicochemical property and physiological activity.The denaturation of protein is usually irreversible , therefore protein, once being denaturalized, their natural property may not return to original state.

In bio-pharmaceutical industry, long term storage of the protein prepared using recombinant DNA technology in aqueous formulation is logical It is often a difficult task.In order to overcome the stability problem of the protein in aqueous formulation, pass through freeze-drying (freeze-drying) It is prepared for more stable human cytokines product.Freeze-drying prods are usually accompanied by the sterile aqueous media for recovery.Multiple After original, these preparations typically have short effective storage life, are also such as when even if saving under low temperature (for example, 5 DEG C) This.

Since lyophilized preparation has a possibility that inconvenience and mistake for carrying out self-healing program, liquid pharmaceutical formulation is to prepare surely Fixed, the safe and effective treatment main selection of antibody drug.However, the problem of protein therapeutic liquid preparation long-term existence It is rendezvous problem, wherein protein molecule physically sticks together, soluble high-molecular amount aggregation etc. is resulted in, It may cause undesirable immune response after application in patients.

Therefore, it is necessary to provide it is a kind of under refrigeration with enhancing stability and at nominal room temperature have at least in Deng stability anti-human IL-17A monoclonal antibody liquid preparation to protect antibody drug during manufacture, storage and administration From degradation or aggreation, thus a possibility that avoiding coming the inconvenience and mistake of self-healing program.

Summary of the invention

Technical problem to be solved by the present invention lies in providing a kind of liquid preparation of anti-human IL-17A monoclonal antibody, It is stable during manufacture, storage and administration, and is suitble to high concentration (such as subcutaneous administration) application.Specifically, Research through the invention finds to add amino acid in the liquid preparation of the anti-human IL-17A monoclonal antibody of high concentration (especially It is lysine and proline) effectively aggregation can be inhibited to be formed, purpose so as to complete the present invention.

Therefore, the first purpose of this invention is to provide a kind of liquid preparation of the monoclonal antibody of anti-human IL-17A.

Second object of the present invention is that the liquid preparation for providing the anti-human IL-17A monoclonal antibody is preparing medicine Application in object.

To achieve the goals above, this invention takes following technical solutions:

The first aspect of the invention is to provide a kind of liquid preparation of the monoclonal antibody of anti-human IL-17A, including resists Human il-17 A monoclonal antibody and amino acid.

Wherein, the amino acid is one or both of lysine and proline.Preferably, the amino acid is dried meat ammonia Acid.

Further, the liquid preparation of the monoclonal antibody of the anti-human IL-17A further includes sugar.Preferably, the sugar For mannitol, sucrose or trehalose.

Further, the liquid preparation of the monoclonal antibody of the anti-human IL-17A further includes surfactant and buffering Agent.Preferably, the surfactant is polysorbate20, polysorbate80 or PLURONICS F87, and the buffer is Histidine buffer, citrate buffer agent and succinate buffers.

Further, the liquid preparation of the monoclonal antibody of the anti-human IL-17A includes following components:

(a) the anti-human IL-17A monoclonal antibody of 25-150mg/ml,

(b) buffer of 10-50mM,

(c) sugar of 50-300mM,

(d) surfactant of 1-20mg/ml,

(e) amino acid of 10-50mM, and

Wherein the pH of the preparation is 5.0-6.0.

It is furthermore preferred that the liquid preparation of the monoclonal antibody of the anti-human IL-17A includes following components:

(a) the anti-human IL-17A monoclonal antibody of 25-150mg/ml,

(b) histidine buffer of 10-50mM,

(c) mannitol of 50-300mM,

(d) polysorbate20 of 1-20mg/ml,

(e) lysine or proline of 10-50mM, and

Wherein the pH of the preparation is 5.0-6.0.

Most preferably, the liquid preparation of the monoclonal antibody of the anti-human IL-17A includes following components:

(a) the anti-human IL-17A monoclonal antibody of 150mg/ml,

(b) histidine buffer of 20mM,

(c) mannitol of 250mM,

(d) polysorbate20 of 8mg/ml,

(e) lysine or proline of 20mM, and

Wherein the pH of the preparation is 5.8.

Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody of the anti-human IL-17A such as SEQ ID Shown in NO:2, the amino acid sequence of light chain variable region is as shown in SEQ ID NO:4.

The second aspect of the invention is that the liquid preparation for providing the monoclonal antibody of the anti-human IL-17A is used for The application of the drug of immune-mediated inflammatory reaction is treated in preparation.

Aforesaid liquid preparation is the composition comprising anti-human IL-17A monoclonal antibody, is given to animal including people After medicine, the inflammatory reaction effect that anti-immunity mediates is obvious.Specifically, prevention to immune-mediated inflammatory reaction and/or controlling It treats effectively, can be used as anti-inflammatory medicaments use.

Signified immune-mediated inflammatory reaction of the invention, including but not limited to: psoriasis, rheumatoid arthritis, silver Bits property arthritis, ankylosing spondylitis, multiple sclerosis, asthma, uveitis, Behcet uveitis, xerophthalmia, it is chronic from Hair property nettle rash.Other than above-mentioned inflammation related disease, it may also be used for multiple sclerosis, Crohn disease, colitis are exedens Colitis, systemic loupus erythematosus, graft versus host disease(GVH disease) etc..Here it will not enumerate.

The inflammatory reaction drug that the so-called anti-immunity of the present invention mediates refers to inhibition and/or treats immune-mediated inflammation Disease reaction drug, may include concomitant immunity mediate inflammatory reaction related symptoms development delay and/or these symptoms it is tight The reduction of weight degree, it further comprises the mitigation of already present inflammatory reaction simultaneous phenomenon and prevents going out for other symptoms It is existing, it further include the transfer for reducing or preventing inflammatory reaction.

The composition of anti-human IL-17A monoclonal antibody gives medicament when to animal administration including people in the present invention It measures age because of patient and weight, disease traits and seriousness and administration route and different, the result of zoopery can be referred to With various situations, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 1-1800mg/ days.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

Beneficial effects of the present invention: the present invention is provided a kind of comprising amino acid by the optimization to sugar and amino acid The liquid preparation of anti-human IL-17A monoclonal antibody, it is preferred that it include lysine and proline, it is furthermore preferred that comprising proline, It is during manufacture, storage and administration it is stable, at normal temperature keep at least a year stabilization, have lower aggregation, The step that rebuilds of lyophilized preparation is needed not move through, and is suitble to high concentration (such as subcutaneous administration) application.This hair The liquid preparation of bright stable anti-human IL-17A monoclonal antibody can effectively apply to treat immune-mediated inflammatory reaction Drug preparation in.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.Room temperature as described in the examples be this field routine room temperature, generally 10~30 DEG C.

Being prepared and obtained according to CN201510407739.9 the method can comprising the heavy chain limited of SEQ ID NO:1 and 2 Become the anti-human IL-17A monoclonal antibody of the humanization of the light chain variable region of area and SEQ ID NO:3 and 4 restrictions.

In following embodiment, stability test, related biological is examined to carry out according to the specification of Chinese Pharmacopoeia.

SDS-PAGE[assembles (non-reduced) and fragmentation (reduction)]

SDS-PAGE is used as a kind of analytical technology, can isolate free sum from native protein according to molecular weight The type of high molecular weight.Due to being inclined in the case where the high molecular weight protein of high concentration with high aggregation, non-reduced SDS- PAGE is used to evaluate covalent aggregation.Due between antibody structure light and weight chain, including there are many disulfide bond for hinge area, also The fragmentation of protein is checked under former SDS-PAGE.

SE-HPLC (aggregation and fragmentation)

With size exclusion chromatography protein isolate matter and its relative substance (based on their size), above-mentioned technology is for inspection The aggregation and fragmentation for surveying monoclonal antibody are useful.

In addition to especially indicating, the reagents and materials used in the present invention are commercially available in following embodiment.

Any those skilled in the art will be understood that the group for the various components being included in composition Conjunction can be completed in any suitable order, that is, it can add first, in centre addition or in finally addition buffer, and And also that can add first, in centre addition or in finally addition surfactant etc..Equally, those skilled in the art It will be understood that some in these chemical substances are incompatible in certain combinations, and therefore, they can be easy Ground is that compatible different chemical substances replace with similar quality but in relation to mixture.

Influence of 1 sugar of embodiment to liquid preparation stability

The anti-human IL-17A monoclonal for adding different types of sugared (sucrose, trehalose, mannitol) by comparative analysis is anti- The aggregation situation of body fluid body preparation during storage, to analyze influence of the different sugar to preparation stability.The liquid system Agent also contains other according to the excipient gone out given in table 1.Aggregation, which mainly passes through SDS-PAGE and SE-HPLC method, to be commented It is fixed.Pharmaceutical formulation and result difference are as shown in Table 1 and Table 2.

Table 1, the anti-human IL-17 monoclonal antibody liquid preparation containing different sugar

The SE-HPLC result of table 2, anti-human IL-17 monoclonal antibody liquid preparation containing different sugar

By the result of table 2 as it can be seen that addition different types of sugared (sucrose, trehalose and mannitol) is mono- for anti-human IL-17A The aggregation formation of clonal antibody liquid preparation has no significant effect.The liquid preparation containing mannitol (F3) is selected to carry out subsequent Research.

Influence of 2 agglutination inhibitor of embodiment to liquid preparation stability

Different types of amino acid (aspartic acid, lysine, alanine, phenylalanine, dried meat ammonia are added by comparative analysis Acid) anti-human IL-17A monoclonal antibody liquid preparation aggregation situation during storage, to analyze different aminoacids pair The influence of preparation stability.The liquid preparation also contains other according to the excipient gone out given in table 3.Aggregation is main It is evaluated by SDS-PAGE and SE-HPLC method.Pharmaceutical formulation and result difference are as shown in Table 3 and Table 4.

Table 3, the anti-human IL-17 monoclonal antibody liquid preparation containing different agglutination inhibitors

The SE-HPLC result of table 4, anti-human IL-17 monoclonal antibody liquid preparation containing different aminoacids

By the result of table 4 as it can be seen that compared with the anti-human IL-17A monoclonal antibody liquid preparation (F3) for not adding amino acid, Addition amino acid effectively can inhibit aggregation to be formed.However, different types of amino acid is anti-for anti-human IL-17A monoclonal The aggregation formation of body fluid body preparation is affected.Specifically, with contain aspartic acid (F5), alanine (F7) and phenylalanine (F8) liquid preparation is compared, and the liquid preparation containing lysine (F6) and proline (F9) has less aggregation.It is more excellent Choosing, the liquid preparation containing proline (F9) has better stability, therefore proline is proved to for preventing anti-human IL- The aggregation of 17A monoclonal antibody liquid preparation is more effective.

The long-term stable experiment of 3 liquid preparation of embodiment

The liquid preparation of anti-human IL-17A monoclonal antibody is prepared according to the formula of table 5, then -20 DEG C, 5 ± 3 DEG C and It stores at 25 ± 2 DEG C 1 month, 3 months, 6 months and after 12 months, is analyzed by SE-HPLC and SDS-PAGE.As a result As shown in table 6.

The liquid preparation of table 5, anti-human IL-17A monoclonal antibody

Serial number	Ingredient	Concentration
			1	Anti-human IL-17A monoclonal antibody	150mg/ml
2	Histidine buffer	20mM
			3	Mannitol	250mM
4	Proline	20mM
			5	Polysorbate20	8mg/ml
6	pH	5.8

Table 6, anti-human IL-17A monoclonal antibody liquid preparation SE-HPLC result

By the result of table 6 as it can be seen that the liquid of the anti-human IL-17A monoclonal antibody comprising 150mg/ml high concentration of the invention Body preparation stability is preferable, can even if still having at least a year preferable stability (aggregation lower than 5%) at normal temperature Meet stability requirement when storage, transport, administration.

Heretofore described anti-human IL-17A monoclonal antibody can also be the Cosentyx of commercialization (secukinumab, Novartis) and Taltz (Ixekizumab, gift come).

It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

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Claims (10)

1. a kind of liquid preparation of the monoclonal antibody of anti-human IL-17A, which is characterized in that anti-including anti-human IL-17A monoclonal Body and amino acid.

2. the liquid preparation of the monoclonal antibody of anti-human IL-17A as described in claim 1, which is characterized in that the amino acid For one or both of lysine and proline.

3. the liquid preparation of the monoclonal antibody of anti-human IL-17A as described in claim 1, which is characterized in that further include sugar.

4. the liquid preparation of the monoclonal antibody of anti-human IL-17A as claimed in claim 3, which is characterized in that the sugar is sweet Reveal alcohol, sucrose or trehalose.

5. the liquid preparation of the monoclonal antibody of anti-human IL-17A as described in claim 1, which is characterized in that further include surface Activating agent and buffer.

6. the liquid preparation of the monoclonal antibody of anti-human IL-17A as claimed in claim 5, which is characterized in that the surface is living Property agent be polysorbate20, polysorbate80 or PLURONICS F87, the buffer be histidine buffer, citric acid Salt buffer agent and succinate buffers.

7. the liquid preparation of the monoclonal antibody of anti-human IL-17A as described in claim 1, which is characterized in that including with the following group Point:

(a) the anti-human IL-17A monoclonal antibody of 25-150mg/ml,

(b) buffer of 10-50mM,

(c) sugar of 50-300mM,

(d) surfactant of 1-20mg/ml,

(e) amino acid of 10-50mM, and

Wherein the pH of the preparation is 5.0-6.0.

8. the liquid preparation of the monoclonal antibody of anti-human IL-17A as claimed in claim 7, which is characterized in that including with the following group Point:

(a) the anti-human IL-17A monoclonal antibody of 25-150mg/ml,

(b) histidine buffer of 10-50mM,

(c) mannitol of 50-300mM,

(d) polysorbate20 of 1-20mg/ml,

(e) lysine or proline of 10-50mM, and

Wherein the pH of the preparation is 5.0-6.0.

9. the liquid preparation of the monoclonal antibody of anti-human IL-17A as claimed in claim 8, which is characterized in that including with the following group Point:

(a) the anti-human IL-17A monoclonal antibody of 150mg/ml,

(b) histidine buffer of 20mM,

(c) mannitol of 250mM,

(d) polysorbate20 of 8mg/ml,

(e) lysine or proline of 20mM, and

Wherein the pH of the preparation is 5.8.

10. the liquid preparation of the monoclonal antibody of anti-human IL-17A as claimed in any one of claims 1-9 wherein, feature exist In the amino acid sequence of the heavy chain variable region of the monoclonal antibody of the anti-human IL-17A is as shown in SEQ ID NO:2, light chain The amino acid sequence of variable region is as shown in SEQ ID NO:4.