UA123847C2 - Liquid formulation of anti-tnf alpha antibody - Google Patents

Abstract

The present invention relates to a liquid formulation of an anti-TNF alpha antibody, particularly adalimumab.

Description

(Technology industry)

The invention relates to a liquid composition of an anti-TME antibody and, in particular, to a liquid composition of adalimumab.

ITechnical level

Tumor necrosis factor-alpha (TME-α) is a cytokine produced by various cell types such as monocytes, macrophages, etc., upon stimulation by endotoxin, etc. TME-α is the main mediator of the main inflammatory, immunological and pathophysiological reactions, which activates TME receptors and causes responses, such as T-cell activation, thymocyte proliferation, etc.

M., her a. (1995) Sei, 83: 793-802).

Adalimumab is a recombinant human immunoglobulin 1, monoclonal antibody that inhibits TME-induced immune responses by selectively binding to TME-a and mimo.

Adalimumab was developed by VAR Viogezeagspy Sogrogayop in 1993 and approved by AB

And abogasgiezh for sale as a treatment for rheumatoid arthritis. Adalimumab is currently marketed under the name NOMICA?, and after receiving approval for its marketing as a therapeutic agent for the treatment of rheumatoid arthritis, NOMICA? used to treat Crohn's disease, ankylosing spondylitis, psoriatic arthritis, nonspecific ulcerative colitis, etc.

Adalimumab is the first fully human antibody developed as a drug.

Adalimumab was developed using phage display technology and has increased affinity by inducing a mutation in SOC. Adalimumab, also referred to as O2E7, consists of 1,330 amino acids and has a molecular weight of approximately 148 kDa (US Pat. No. 6,090,382).

Adalimumab is a TME-a inhibitor that binds to TME and blocks TME-induced responses by preventing TME from interacting with cell surface TME receptors (ie, p55 and p7b).

At the same time, antibody-based drugs are a type of protein drug, and physical and chemical denaturation may occur due to various factors. Protein denaturation can be chemical denaturation, such as oxidation, deamidation, and isomerization, or structural denaturation, such as fragmentation or aggregation. When a protein is denatured, it is possible that the protein may lose its intrinsic pharmacological activity and thus

In this way, to cause unnecessary immune reactions ip mimo as a side effect. When an antibody is fragmented, its binding affinity or retention time in the body changes, thus affecting the pharmacological activity of the antibody. In addition, studies have shown that fragmented antibodies can cause antibody aggregation. In addition, pharmacological activity may be reduced due to aggregation. According to research, as a result of comparing commercial interferon-beta products, it was confirmed that those products that have a high content of aggregates and particles are likely to have a high percentage of neutralizing antibodies in the body (Vagpaga et al., 2013, 9). React. 5 102: 915). If neutralizing antibodies are produced intravenously, the neutralizing antibodies bind to the protein drug when the drug is injected into the body, thereby affecting the safety, pharmacology, and pharmacokinetics of the protein drug. In addition, denaturation and aggregation of epoetin alfa have also been shown to cause increased immunogenicity of epoetin alfa medicinal products. Accordingly, in the case of protein drugs, it is very important to obtain protein drugs in appropriate compositions, so that they do not lose their physiological activity during the storage period, and also that the proteins are not aggregated or formed into particles.

Accordingly, active research on compositions for various protein medicines continues.

The goal of protein composition research is to find the optimal combination by appropriately mixing several additives, taking into account the properties of each product, so that the product can be stably stored until it is administered to the patient. The main purpose of adding additives is to stabilize the proteins and control the physical properties of the mixed materials. Additives can be divided into surfactants, stabilizers, preservatives, buffers, isotonic agents, etc. depending on their purpose and characteristics. In the case of antibody-based drugs, a larger amount of protein is administered compared to other protein drugs in such a way as to achieve effective therapeutic effects. In the case of subcutaneous injection, it is important to develop highly concentrated compositions due to the difficulties of delivering a large volume at one time, such as patient pain, retrieval, etc. As the concentration of the protein increases, its intermolecular interaction increases, thus causing problems such as increased aggregation, 60 increased viscosity, gelation, precipitation, etc. Among them, an excessive increase in viscosity complicates production, and also complicates patient administration due to increased injection pressure. Thus, various methods for predicting and reducing the viscosity of a highly concentrated solution of antibodies are currently being studied.

Disclosure)

(Technical problem)

The task of the invention is to create a liquid composition of anti-HME-a antibodies.

Another task of the invention is to create a method for obtaining a liquid composition.

Another task of the invention is to create a method for improving the stability of anti-HME-a antibodies using a composition that contains a stabilizer, a surfactant and arginine.

Another object of the invention is to create a method for improving the stability of an anti-HME antibody using a composition that contains a stabilizer, a surfactant and arginine without a buffer.

ITechnical solution

Further in this document, the invention will be described in more detail.

Meanwhile, each of the explanations and illustrative embodiments disclosed herein may be applied to other explanations and illustrative embodiments.

That is, all combinations of various factors disclosed in this document are within the scope of the invention. Furthermore, the scope of the invention should not be limited to the specific disclosure presented herein below.

In addition, one of ordinary skill in the art will be able to recognize or confirm, using no more than routine experimentation, the appropriate number of equivalents of the specific embodiments of the invention described herein. In addition, such equivalents, as intended, are included in the invention.

To achieve the above objectives, one aspect of the invention relates to a liquid composition of an anti-TME-a antibody.

As used herein, the term "anti-TME-a antibody" refers to an antibody that binds to TME-a and regulates its biological activity. More specifically, the antibody can bind to TME-a and inhibit binding between TME-c and its receptors, thereby

By inhibiting signaling with the help of TME. In addition, the anti-TME antibody may be a monoclonal antibody.

The anti-TME-a antibody can be in the form of a full-length antibody or an antibody fragment containing its antigen-binding site, but the anti-HTME-y4 antibody is not specifically limited to them.

More specifically, the anti-TME antibody may be recombinant human immunoglobulin

A C1 monoclonal antibody, and even more specifically, can be adalimumab. Information about adalimumab can be readily obtained by those skilled in the art from known databases.

The antibody can be obtained using recombinant DNA technology using a mammalian cell expression system, but the method of obtaining is not limited to this.

The antibody can be contained in a liquid composition according to the invention in a therapeutically effective amount. In a specific embodiment, the antibody may be present at a concentration of from 1 mg/ml to 250 mg/ml, in particular from 20 mg/ml to 200 mg/ml, more specifically from 50 mg/ml to 200 mg/ml, and even more specifically at a concentration of 50 mg/ml, 100 mg/ml, or 130 mg/ml, but the concentration of the antibody is not specifically limited thereto.

The liquid composition of the invention may contain a stabilizer, a surfactant, and arginine, in addition to an anti-TME-sa antibody. The liquid composition can be a composition in the form of a solution capable of stably storing the anti-TME antibody.

In particular, the stability of an anti-TME-α antibody can be measured using any protein stability assay well known in the art. Stability can be measured at a selected temperature for a selected time. For a rapid test, the composition can be stored at a higher or "elevated temperature" (eg, at 40 "C for 2 weeks to 1 month or more), at which the time-dependent stability value is measured.

In the invention, "ensuring the stability of the anti-TME-α antibody" means that the loss of the active ingredient under certain storage conditions (in particular, at a certain temperature) during a certain period of time is less than a given amount (for example, less than 10 95 ). Typically, when the residual value of anti-TME-sa antibodies in the composition is 90 95 or more, especially about 92 95 or more at 5:53 "C for 2 years, at 25:22 "C for 6 months, or 402 "C for 1 to 2 months, these compositions can be interpreted as being stable.

The stabilizer, which should be contained in the liquid composition according to the invention, can be a polyol, an amino acid, or a combination thereof. The amino acid can be an amino acid other than arginine.

In particular, the stabilizer can be 1) one type of polyol; 2) a combination of one type of polyol and one type of amino acid; 3) a combination of one type of polyol, the first amino acid and the second amino acid; 4) a combination of the first polyol and the second polyol; 5) a combination of the first polyol, the second polyol and one type of amino acid; b) a combination of the first polyol, the second polyol, the first amino acid and the second amino acid; or 7) one type of amino acid.

More specifically, the polyol can be mannitol, sucrose, trehalose, PEG, or a combination thereof, and more specifically, sucrose, trehalose, PEG, or a combination thereof. In particular, the PEG may be PEG400 or PEG4000, but is not specifically limited thereto. In the above preparation, the polyol can be present in a concentration from 0.1 mg/ml to 100 mg/ml.

More specifically, the amino acid other than arginine can be glycine, leucine, isoleucine, phenylalanine, or proline. In the above composition, the amino acid may be present in a concentration of 1 mM to 300 mM.

In addition, as used herein, the term "amino acid" also includes all those, in the form of analogs, solvates, hydrates, stereoisomers, and pharmaceutically acceptable salts of the corresponding amino acid, which exhibit essentially the same effect.

As used herein, the term "pharmaceutically acceptable salt" includes pharmaceutically acceptable inorganic acids, organic acids, or salts derived from bases.

Examples of suitable acids may include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid , acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Examples of salts derived from appropriate bases may include alkali metal salts such as sodium, potassium, etc.; alkaline earth metal such as magnesium, etc.; ammonium, etc.

Also, as used herein, the term "solvate" means that

An amino acid or its salt forms a complex with a solvent molecule.

More specifically, the stabilizer may be a stabilizer selected from the group consisting of () sucrose or trehalose, (ii) PEG having an average molecular weight of 200 to 600, or

PEG, which has an average molecular weight of 1000 to 8000; (ii) glycine or leucine; and (im) combinations of at least two of (ii) to (ii), but the stabilizer is not specifically limited to them.

In a more specific embodiment, the stabilizer may be a stabilizer selected from the group consisting of 1) any one of sucrose, trehalose, and PEG400; 2) combinations of sucrose or trehalose with glycine or leucine; 3) combinations of sucrose or trehalose with glycine and leucine; 4) combinations of sucrose or trehalose with PEG4000; 5) combinations of sucrose or trehalose with PEG4000 and glycine; b) combinations of sucrose or trehalose with

PEG4000 and leucine; 7) combinations of sucrose or trehalose with PEG4000, glycine and leucine; and 8) glycine, but the stabilizer is not specifically limited thereto.

The surfactant to be contained in the liquid composition according to the invention can be a nonionic surfactant. More specifically, the surfactant can be a polysorbate or a poloxamer.

In particular, the surfactant may be polysorbate 80, polysorbate 20, or poloxamer 188, but the surfactant is not specifically limited thereto.

In the above composition, the surfactant may be present in a concentration of 0.1 mg/ml to 5 mg/ml.

Arginine, which should be contained in the liquid composition according to the invention, can be present in the form of a salt, and more specifically, in the form of a pharmaceutically acceptable salt.

More specifically, arginine may be in the form of arginine hydrochloride, but arginine is not specifically limited thereto.

In the above composition, arginine can be present in a concentration of from 0.1 mM to 200

MM. More specifically, arginine may be present at a concentration of 0.1 mM to 140 mM when the antibody is present in the composition at a concentration of 100 mg/ml, and arginine may be present at a concentration of 0.1 mM to 100 mM when the antibody is present in the composition at a concentration of 50 mg/ml, but the concentration of arginine is not specifically limited to this.

Arginine in the liquid composition according to the invention can be contained as an agent that reduces viscosity.

Due to the fact that it contains arginine, the liquid composition according to the invention can have a viscosity of from about 1 sp to 6 sp, but the viscosity is not specifically limited to this. Viscosity can be measured using various methods known in the art, for example, in the same way as described in Example 1, but the measurement method is not specifically limited to this.

The liquid composition according to the invention may additionally contain an antioxidant.

As used herein, an "antioxidant" may act to inhibit the formation of impurities that may occur as a result of protein oxidation reactions in a dissolved state.

Examples of the antioxidant may include sodium hydrosulfate, ascorbic acid, ascorbyl palmitate, citric acid, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), thioglycerol, propyl gallate, methionine, sodium ascorbate, sodium citrate, sodium sulfide, sodium sulfite, EDTO, and other antioxidants, but antioxidant is not limited to this.

In the above composition, the antioxidant (in particular, methionine) may be present in a concentration of 1 mM to 50 mM, but the antioxidant is not specifically limited to this.

In addition, the liquid composition of the invention may have a pH of 4 to 6, but the pH is not specifically limited thereto.

At the same time, the liquid composition cannot additionally contain a salt or a buffer, but the liquid composition is not specifically limited to this.

In a non-limiting embodiment, the composition containing anti-TME-4 antibody at a concentration of 50 mg/ml or higher may not additionally contain salt and/or buffer.

As confirmed in an embodiment of the invention, a composition according to the invention that does not contain salt and buffer can provide greater stability of the anti-TME antibody to heating, compared to compositions that do not contain salt, buffer, or both. but the composition according to the invention is not specifically limited thereto.

At the same time, if additional to the drug composition with a high concentration of antibodies (for example, a drug in which the antibodies are present at a concentration of 50 mg/ml or higher) or an additional additive is not added to the composition in such an amount that causes the osmotic pressure solution to deviate from an osmotic pressure range similar to that of body fluids, pain during administration can be reduced, thus improving patient comfort.

At the same time, the liquid composition according to the invention can have the following effects, but these

The effect is not specifically limited to this.

A liquid composition containing arginine according to the invention can demonstrate a relatively low content of high molecular weight (HMUM) products due to the inhibition of anti-HME-a antibody proteins compared to a composition that does not contain arginine; talaba may include a relatively low amount of acidic variants of the antibody due to inhibition of the production of acidic variants of the antibody, compared to a composition that does not contain arginine. In particular, the liquid composition containing arginine according to the invention can have the effect of reducing the antibody produced by denaturation and/or the effect of reducing aggregation and particle formation in response to certain physical stresses.

The composition in the form of a solution containing arginine according to the invention may additionally contain a preservative. A preservative refers to a compound that is added to a pharmaceutical composition to act as an antimicrobial agent. Examples of the preservative may include benzalkonium chloride, benzethonium, chlorhexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercury nitrate, thimerosal, benzoic acid, etc., but the preservative is not limited to hereby. These preservatives can be used alone or in any combination of at least two types.

The liquid composition according to the invention can be in the form of a pharmaceutical composition.

In addition, the liquid composition according to the invention may contain various pharmaceutically acceptable carriers, in addition to the ingredients described above.

The compositions of the invention can be used for the prevention or treatment of rheumatoid arthritis, psoriasis, psoriatic arthritis, axial spondyloarthritis (for example, ankylosing spondylitis, severe axial spondyloarthritis without radiological signs of ankylosing spondylitis), vasculitis, Alzheimer's disease, ulcerative colitis, Behcet's enteritis, hidradenitis suppurativa, uveitis , juvenile idiopathic arthritis, pediatric psoriasis, or Crohn's disease (including but not limited to adult Crohn's disease and pediatric Crohn's disease).

The compositions of the invention may be administered by oral administration or parenteral administration, including, but not limited to, subcutaneous, intramuscular, intraperitoneal, intrasternal, transdermal, and intravenous injection and infusion.

Another aspect of the invention relates to a method of obtaining a liquid composition of anti-TME-a antibody, which includes mixing anti-TME-a antibody, stabilizer, surfactant and arginine with each other.

The above terms are the same as described above. Furthermore, it is clear that all specific embodiments of these terms will also apply to an aspect.

Another aspect of the invention relates to a method of improving the stability of an anti-TME-a antibody using a composition containing a stabilizer, a surfactant and arginine.

The above terms are the same as described above. Furthermore, it is clear that all specific embodiments of these terms will also apply to an aspect.

Another object of the invention is to create a method for improving the stability of an anti-HTME-a antibody using a composition that contains a stabilizer, a surfactant and arginine without a buffer.

The above terms are the same as described above. Furthermore, it is clear that all specific embodiments of these terms will also apply to an aspect.

(Predominant effects of the invention)

The liquid preparation of anti-TME-x antibodies according to the invention, in particular, the liquid composition of adalimumab, can reduce the formation of co-products of adalimumab during storage, which allows long-term storage. In addition, the pharmacological effect of adalimumab can be stably preserved for a long period of time due to the prevention of denaturation and aggregation, which is caused in response to physical stresses during the manufacturing and transportation processes.

Accordingly, the liquid composition according to the invention can be effectively used in the therapeutic field related to the pharmacological effectiveness of adalimumab.

I Brief description of the drawings)

Figure 1 shows the viscosity of each sample in example 1, according to additive compositions.

Figure 2 shows the number of particles in each sample and their placebo in Example 7, before and after passing through the pump.

(Detailed description of the invention)

Further in this document, the invention will be described in more detail with reference to the following examples. However, these examples are given for illustrative purposes only and the invention is not limited

With these examples. "Example 1"

Viscosity-reducing effect of adalimumab solution according to additives and confirmation of stability

In order to confirm the additives that will be used to prepare the liquid preparation of adalimumab, composition 1 was obtained with the following components: sucrose (55 mg/ml), methionine (5 tM), polysorbate 80 (1 mg/ml), and adalimumab (100 mg /ml) and with a pH of 5.2.

In addition, compositions 2-13 were obtained by further adding each of arginine hydrochloride, lysine hydrochloride, leucine, isoleucine, phenylalanine, glutamic acid, glycine, proline, alanine, sodium chloride, calcium chloride, and magnesium chloride to composition 1, and in The viscosity of each composition was measured using a tukos apparatus (Kpeozepvze Ips.). The types and concentrations of additives added to the compositions and the viscosity of each composition are shown in Table 1 below and Figure 1.

Table 1

Composition! ///Ї77777777111111111111111-1111111111111111111111113231С

Regarding the viscosity for each composition listed in Table 1, the viscosity for composition 1 consisting of sucrose, methionine, polysorbate 80 and adalimumab was 3.23. In comparison, the viscosity of compositions containing amino acids such as leucine, isoleucine, phenylalanine, glycine and proline ranged from 3.20 to 3.28, thus not showing any significant difference from composition 1. In contrast, in cases where arginine hydrochloride, lysine hydrochloride, glutamic acid, sodium chloride, calcium chloride, magnesium chloride, etc. were added, the viscosity was confirmed to be in the range of 2.94 to 3.14, which shows a decrease compared to composition 1.

To compare the stability of each formulation, each formulation was sterilized by filtration using a filter (pore size: 0.2 μm), filled into glass syringes (volume: approximately 1.0 mL) in an amount of 0.4 mL each, capped, and stored at 40 °C for 2 months. After storage, each sample was analyzed by 5E-HPLC to analyze the content of high molecular weight impurities (HMU) (eg, oligomers, aggregates, etc.) and low molecular weight impurities (I MU) (ie, fragments of adalimumab molecules).First, the 5E-HPLC results of the reduced viscosity compositions (Compositions 2, C, 7, 11, 12 and 13) and Composition 1 as a control group are presented in Table 2 below In addition, the ZE-HPLC results of compositions without any significant changes in viscosity (compositions 4, 5, 6, 8, 9 and 10), and composition 1 as a control group are presented in Table C below.

Table 2

SHY S an -ishny months

Table Z of nn months (Composition170 | 0.34 | 056 | 090 | 124 | 484 | 608

With respect to Table 2 above, the NMMU, IGMMU, and total storage impurity amounts were shown to be close among the compositions. However, the total amount of impurities, after storage at 40 "C for 2 months, was 7.10 95 in composition 11 containing sodium chloride, 7.38 96 in composition 12 containing calcium chloride, and 7.42 95 in composition 13 containing magnesium chloride, thus showing a significant increase compared to 6.59 95 of composition 1 (i.e., the control group), respectively.In contrast, in the cases of compositions 2, 3, and 7, which included arginine hydrochloride, lysine hydrochloride, and glutamic acid, respectively, the total amounts of impurities after storage at 40 "C for 2 months were shown to be in the range from 6.45 95 to 6.61 95, thus maintaining the levels of the total amount of impurities around to 6.59 95 composition 1 (ie, control group).

With reference to Table C above, in the case of compositions to which amino acids such as leucine, isoleucine, phenylalanine, glycine, proline, etc. were added, the total amounts of impurities after storage at 40°C for 2 months were shown to be in the range of 5 .94 95 to 6.17 95, thus showing a significant decrease compared to the 6.59 95 of Composition 1 (ie,

control group). Based on these results, it was confirmed that amino acids such as leucine, isoleucine, phenylalanine, glycine, proline, etc. can contribute to the stability of adalimumab. "Example 2"

Stability rating 1 according to the content of arginine hydrochloride

In order to evaluate the stability of adalimumab compositions according to the content of arginine hydrochloride, the compositions were prepared as follows.

Composition 14 was prepared in such a way that it contained sucrose (55 mg/ml), methionine (5

MM), polysorbate 80 (1 mg/ml) and adalimumab (100 mg/ml). Compositions 15 and 16 were obtained by adding arginine hydrochloride (20 mM) and arginine hydrochloride (40 mM) to composition 14, and loaded into 1 ml glass syringes in the amount of 0.4 ml, respectively. Each syringe was stored at 40 "C for 2 months and then subjected to 5E-HPLC stability analysis. The composition of each composition and the content of NMMU before and after storage are shown in Table 4 below.

Table 4. Before storage at |After storage at months months

Sucrose (55 mg/ml), Methionine (5

Composition 14 MM), Polysorbate 80 (1 mg/ml), and 0.17 1.00

Arginine hydrochloride (0 mM)

Sucrose (55 mg/ml), Methionine (5

Composition 15 MM), Polysorbate 80 (1 mg/ml), and 0.17 0.682

Arginine hydrochloride (20 mM)

Sucrose (55 mg/ml), Methionine (5

Composition 16 µM), Polysorbate 80 (1 mg/ml), and 0.16 0.682

Arginine hydrochloride (40 mM

Regarding the ZE-HPLC results in Table 4, when the content of arginine hydrochloride was increased from 0 mM (Composition 14) to 20 mM (Composition 15) and 40 mM (Composition 16), the amount

NMUM in samples before storage was close, in the range from 0.16 95 to 0.17 95. However, the content

NMUM after storage at 40 "C for 2 months was 1.00 95 in composition 14 (which does not contain arginine hydrochloride), and 0.82 95 in compositions 15 and 16 (containing 20 mM arginine hydrochloride and 40 mM arginine hydrochloride, respectively ), thus showing that formulations containing 20 mM arginine hydrochloride and 40 mM arginine hydrochloride decreased levels of

NMUM in comparison with the composition that does not contain arginine hydrochloride. Accordingly, it was confirmed that arginine hydrochloride has a preventive effect against the aggregation of adalimumab. "Example 3"

Comparison of the formation of variants that differ in antibody charges according to the content of arginine

In order to compare the levels of formation of variants that differ in charges according to the content of arginine, a composition containing arginine and a composition without

Z arginine, stored at 40 "C for 1 month, and the signs of formation of charge-differentiated variants were compared using cation-exchange HPLC. Each of the compositions and content of charge-differentiated variants before and after storage is shown in Table 5 below.

Table 5

Composition of 1 month for 1 month

Sucrose (55 mg/ml), Methionine (5 MM),

A Polysorbate 80 13.36 /|70.77112.44 3.44 33.39 |51.58110.70 4.33 (1 mg/ml), and

Adalimumab (100 mg/ml)

Sucrose (55 mg/ml), Methionine (5 mm)

Polysorbate 80 (1 mg/ml), iz3y5 |71m4|1241) 330 31.81 |BaBI1Я6| 5

Adalimumab (100 mg/ml), and arginine hydrochloride (20

mm)

Formulation A was formulated to contain sucrose (55 mg/ml), methionine (5 mM), polysorbate 80 (RB8O: 1 mg/ml) and adalimumab (100 mg/ml), and formulation B was formulated to contain it additionally contained arginine hydrochloride (20 mM) in composition A. The content of acid variants of these two compositions before storage at 40 "C for 1 month were similar. When comparing the data of the two compositions after storage, it was confirmed that composition B, which contains arginine hydrochloride, had a lower content of acidic variants of the antibody and a higher content of KO compared to composition A.

Accordingly, it was confirmed that arginine hydrochloride reduces the formation of acid variants of adalimumab. "Example 4"

Compositions according to the types of surface-active substances and comparison of the stability of these compositions

In order to compare the stability of liquid compositions of adalimumab according to the types of surface-active substances, compositions having the following compositions were obtained.

As in composition 14 of example 2, composition 17 was prepared so that it contained sucrose (55 mg/ml), methionine (5 mM), polysorbate 80 (1 mg/ml), and adalimumab (100 mg/ml). In addition, compositions 18 and 19 were obtained using a change in the type of surfactant to polysorbate 20 and poloxamer 188, while the fixed amount of surfactant was the same.

Each composition was sterilized by filtration, filled into each 1 ml glass syringe in the amount of 0.4 ml, and stored at 40 "C for 1 month. The amount of NMUM and GMUM impurities in the samples before and after storage was analyzed using ZE-HPLC. The composition of the compositions 17-19 and the content by 5E-HPLC of these compositions before and after storage at 40 C for 1 month was analyzed, and the results are shown in table b below.

Table 6

Before storage at 40 "C | After storage at 40 "C for 1 month for 1 month

NMUU | And MMU | Total/| State Medical University In general, St

Sucrose (55mg/ml), . Methionine (5mM),

Composition 17 Polysorbate 80 (mg/ml), 0.14 0.37 0.51 0.46 2.05 2.52 pH52

Sucrose (55mg/ml), . Methionine (5mM),

Composition 18 Polysorbate 20 (mg/ml), 015 0.38 0.53 0.45 2.04 2.43 pH52

Sucrose (55mg/ml),

Methionine (5mM),

Composition 19 | Poloxamer 188 0.15 0.37 0.53 0.44 1.99 2.43 (mg/ml), pH52

Regarding the results from Table b, the content of NMMUU and GMUM before and after storage at 40 C for 1 month did not change significantly depending on the types of surface-active substances.

That is, the stability of the composition containing polysorbate 80, the composition containing polysorbate 20, and the composition containing poloxamer 188 were similar to each other.

Accordingly, it was confirmed that the effects of polysorbate 80, polysorbate 20, and poloxamer 188 on the stability of adalimumab were similar to each other. "Example 5"

Compositions according to types of polyols and comparison of the stability of these compositions

In order to compare the stability of liquid compositions of adalimumab according to the types of polyol, compositions having the following compositions were obtained.

As in formulation 14 of example 2, formulation 20 was prepared to contain sucrose (55 mg/mL), methionine (5 mM), polysorbate 80 (1 mg/mL), and adalimumab (100 mg/mL) (so same as composition 17 in example 4). In addition, compositions 21-23 were obtained by changing the type of polyol to trehalose, PEG400, and PEG4000, while the fixed total amount of polyol was the same.

Each composition was sterilized by filtration, filled into each 1 ml glass syringe in the amount of 0.4 ml, and stored at 40 "C for 1 month. The amount of NMUM and GMUM impurities in the samples before and after storage was analyzed using 5ZE-HPLC.

The composition of compositions 20-23 and the content by BE-HPLC of these compositions before and after storage at "C" for 1 month were analyzed, and the results are shown in Table 7 below.

Table 7

Before storage at 40 "C | After storage at 40 "C for 1 month for 1 month

NMUU | And MMU | General | NMMU | And MMU | General

Cho Cho Cho Cho Cho Cho

Sucrose (55 mg/ml),

Compositions - | Methionine (5 mM), and cation 20 Polysorbate 80 (1 mg/ml, pH 1 0097 | b 046 |205| 252 5.2

Trehalose (55 mg/ml),

Compositions - | Methionine (5 mM), titration 21 Polysorbate 80 (1 mg/ml), pH 07 0096) 050 046203) 249 5.2

Compositions - PEG400 (55 mg/ml), Methionine (5 mM), and Polysorbate 80 (1 0.16 | 0.38 0.54 0.62 | 2.02 2.63 tion 22 mg/ml), pH 5.2

---- out of January for 1 month for 1 month Tue TVO i

Cho Cho Cho Cho Cho Cho she (ven o|ny| oyu |ov z» oyu - (5 mM), and Polysorbate 80 (1 0.17 | 0.37 0.53 0.85 | 2.05 2.90 concentration 23 mg/ml), pH 5.2

Regarding the results from Table 7 above, it can be seen that the content of NMMU and GMMU varies significantly depending on the types of polyols before and after storage at 40 "C for 1 month.

The content of NMUM and GMMU impurities of all compositions was similar to each other before storage at 40 "C for 1 month. However, after storage at 40 "C for 1 month, compositions 20-22, which contained sucrose, trehalose and PEG400, respectively, showed relatively low content

NMUM in comparison with composition 23, which contained PEG4000. The content of !MMUU, as shown, was similar among the given compositions. Accordingly, it was confirmed that there is a difference in stability between the compositions depending on the types of polyols used, and among them, sucrose and trehalose are shown to be more effective in improving stability compared to other types of polyols. "Example b"

Comparison of stability between compositions consisting of polyol, arginine, methionine, surfactant, and additional stabilizer, and composition NOMIVA?

Samples were obtained using different compositions by changing the types of surfactants and polyols, the presence/absence of an additional stabilizer, the content of arginine hydrochloride, etc., in a composition where 5 mM methionine was used as a stabilizer, and a composition corresponding to the commercial preparation Nitiga?x was prepared. These samples were stored at 40 "C, and then subjected to 5E-HPLC analysis to compare the stability among these compositions. The compositions for each of these compositions are shown in Table 8, and the content of impurities before and after storage at 40 "C for 2 months is shown in the table 9.

Table 8

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (0 mM), polysorbate 80 (1 mg/ml), sucrose (55 mg/ml)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (20 mM), polysorbate 80 (1 mg/ml), sucrose (55 mg/ml)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), polysorbate 80 (1 mg/ml), sucrose (55 mg/ml)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), sucrose (55 mg/ml),

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), sucrose (55 mg/ml), glycine (20 mM)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), sucrose (55 mg/ml), leucine (20 mM)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), sucrose (55 mg/ml), glycine (10 mM), leucine (10

mm)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), trehalose (55 mg/ml)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), trehalose (55 mg/ml), glycine (20 mM)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), trehalose (55 mg/ml), leucine (20 mM)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 34 poloxamer 188 (1 mg/ml), trehalose (55 mg/ml), glycine (10 mM), leucine (10

mm)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), sucrose (27.5 mg/ml), PEG4000 (27.5 mg/ml)

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 36 poloxamer 188 (1 mg/ml), sucrose (27.5 mg/ml), PEG4000 (27.5 mg/ml ), glycine (20 mM

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 37 poloxamer 188 (1 mg/ml), sucrose (27.5 mg/ml), PEG4000 (27.5 mg/ml ), leucine (20 mM)

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM),

C8 poloxamer 188 (1 mg/ml), sucrose (27.5 mg/ml), PEG4000 (27.5 mg/ml), glycine (10 mM), leucine (10 mM)

Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), poloxamer 188 (1 mg/ml), trehalose (27.5 mg/ml), PEG4000 (27.5 mg/ml)

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 40 poloxamer 188 (1 mg/ml), trehalose (27.5 mg/ml), PEG4000 (27.5 mg/ml ), glycine (20 mM

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 1 poloxamer 188 (1 mg/ml), trehalose (27.5 mg/ml), PEG4000 (27.5 mg/ml ), leucine (20 mM)

SHE Adalimumab (100 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), 42 poloxamer 188 (1 mg/ml), trehalose (27.5 mg/ml), PEG4A000 (27.5 mg/ml ), glycine (10 mM), leucine (10 mM)

Adalimumab (50 mg/ml), methionine (5 mM), arginine hydrochloride (40 mM), polysorbate 80 (1 mg/ml), sucrose (55 mg/ml) x Commercial composition NOMICA?E: sodium dihydrogen phosphate dihydrate (0, 86 mg/ml), sodium hydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0.3 mg/ml), citric acid monohydrate (1.3 mg/ml), mannitol (12 mg/ml), sodium chloride (6.16 mg/ml), polysorbate 80 (RBZ80) (1 mg/ml), pH 5.2.

Table 9

Mo. months months 111117 D|NMU (ov) MM (95) |General(95) |NMM/(96) GMUM (96) |General (96).ShCh/ 724 | 077 | 044 | 060 | 00 | 442 | 543 Sh 27. | 077 | 043 | 060 | 085 | 457 | 542 7.29 | about | 044 | 060 | 080 | 455 | 5534 | 07 | 044 | 060 | 0.79 | 454 | 5933 33 | 07 | 043 | 060 | 0.076 | 455 | 53 34 | about | 044 | 060 | 0.076 | 457 | 5933 | ov | 045 | 062 | 099 | 456 | 555 39 | 07 | 043 | 061 | 096 | 452 | 548 741. | 077 | 042 | 059 2 shSh | 099 | 450 | 549 42 | ol | 043 | 061 | 099 | 455 | 555

Mo. months months 1111117 D|NMU (in) MM (95) |General(95) |NMM/ (96) GMUM (96) |General (96).ShCh/

As shown in Table 8, sucrose, trehalose, a combination of sucrose and PEG4000, and a combination of trehalose and PEG4000 were used as polyols, while polysorbate 80 and poloxamer 188 were used as surfactants.

Arginine was used at a concentration of 0 mM, 20 mM, or 40 mM, and to confirm the role of additional additives, compositions were developed using glycine (Su), leucine (em), or a combination of glycine and leucine, and the stability of the resulting compositions adalimumab (100 mg/ml) was compared with the commercial composition NOMIVAE. In addition, regarding the stability of adalimumab (50 mg/ml), the commercial composition of NOMICA? was compared with the composition of composition 44, which is the same as composition 26, except for the adalimumab content.

As a result of the comparison of stability among the compositions given in Table 9, it was confirmed that all preparations had excellent stability in comparison with the commercial composition NOMICAE. Total impurity content before storage ranged from 0.57 to 0.74 and were similar to each other. However, regarding the stability of these compositions after storage at 40 "C for 2 months, it was confirmed that the other compositions had a lower content of adalimumab impurities compared to the commercial composition NOMIVA?, which has adalimumab content of 100 mg/ml (composition 43), and commercial composition NOMIVBA-E, which has adalimumab content of 50 mg/ml (composition 45).

Accordingly, it was confirmed that the compositions according to the invention, which consist of various polyols and surfactants, arginine hydrochloride, and an additional stabilizer, are more stable than the commercial NOMIVA-E compositions with respect to increasing impurities.

In addition, while the total impurity content was about 0.4 95 or higher, in the compositions of Example 5 after storage at 40 "C for 1 month, when PEG 4000 was used as the polyol in Example 5 compared to the compositions when sucrose or trehalose was used as a polyol in the compositions of this example, the total impurity content of adalimumab when sucrose or trehalose was used together with PEG4A000, although the shelf life was 2 months, was shown to be similar to those compositions in which sucrose or trehalose were used alone Accordingly, it has been confirmed that when an additional additive is added to obtain an additional effect such as antioxidant, etc., replacing part of sucrose or trehalose with PEG, which has a higher molecular weight, can be used as a way to maintain osmotic pressure and stability. "Example 7"

Comparative stability assessment of the commercial composition NOMICAF and compositions containing arginine hydrochloride in response to mechanical stress

In order to compare the stability of the commercial composition of NOMICA? and compositions containing arginine hydrochloride in response to mechanical stress, compositions with the following composition were obtained and allowed to pass through a rotary piston pump, and thus the number of particles before and after passing through the pump was evaluated.

In addition, to confirm whether adalimumab-derived particles were measured, placebos were treated with the exception of adalimumab alone in each sample, allowed to pass through a rotary piston pump under the same conditions, and thus the number of particles before and after pump passage was evaluated. To estimate the number of particles, a micro device was used to form an image of the flow of a simple protein. The composition for each sample is presented in Table 10, and the number of particles of each sample and their placebo before and after passing through the pump are shown in Table 11 and Figure 2.

Table 10

Adalimumab (100 mg/ml), sucrose (55 mg/ml), arginine hydrochloride (40 mM), 46 methionine (5 mM), polysorbate 80 (1 mg/ml), pH 5.2

Adalimumab (50 mg/ml), sucrose (55 mg/ml), arginine hydrochloride (40 mM), 47 methionine (5 mM), polysorbate 80 (1 mg/ml), pH 5.2

Adalimumab (50 mg/ml), sodium dihydrogen phosphate dihydrate (0.86 mg/ml), sodium

K. dihydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0.3 mg/ml), lemon ointment. : . 48 acid monohydrate (1.3 mg/ml), mannitol (12 mg/ml), sodium chloride (6.16 mg/ml), polysorbate 80 (RBZV80) (1 mg/ml), pH 5.2 (composition of commercial nomics?

Table 11 11111111 Concentration of particles of fumluYidH//:/SSSSSS:SSSSSS( OS through the pump through the pump through the pump through the pump

She and 17560015

As shown in Table 10, composition 46 and composition 47 have the same additive composition, and the content of adalimumab in composition 46 is 100 mg/ml, and composition 47 is 50 mg/ml. In the case of composition 48, the composition and content of adalimumab, as adjusted, is the same as in commercial NOMICA?E. Each composition was passed through a rotary piston pump, and the number of particles was analyzed before and after passing. As a result, as shown in Table 11 and Figure 2, it was confirmed that Composition 46 had 85.743 particles/ml and Composition 47 had 53.734 particles/ml, and Composition 46 with high adalimumab content had a higher number of particles compared to Composition 47. with low adalimumab.

In contrast, in the case of formulation 48, i.e., the commercial formulation NOMIKAE?, the number of particles that passed through the pump was 150.617 particles/mL, thus showing a higher particle number compared to formulations 46 and 47. Furthermore, the particle concentrations the placebo of all post-pump formulations was 3.618 particles/mL and 7.938 particles/mL, and thus the post-pump particles measured in the adalimumab-containing samples were confirmed to be from adalimumab.

Accordingly, it was confirmed that compositions containing arginine hydrochloride can effectively protect adalimumab in response to mechanical stress, in comparison with the commercial composition NOMICAE. "Example 8"

An experiment on the effect of viscosity reduction depending on the concentration of arginine hydrochloride

In order to investigate the range of arginine concentration at which the viscosity of the adalimumab solution could be reduced when arginine was added to it, the experiments were performed as follows. In the control group, which does not contain arginine hydrochloride (AgoNSII), samples containing adalimumab (100 mg/ml) and polysorbate 80 (1 mg/ml) or adalimumab (50 mg/ml) and polysorbate 80 (1 mg/ml) were received. ml). In the experimental group, samples containing arginine hydrochloride were obtained by gradually increasing the concentration of arginine hydrochloride according to

Due to the addition to each composition of the control group from 20 mM up to 180 mM (final concentration). The pH of all samples was approximately 5.2. The viscosity of the obtained samples was measured using the TucoOs device from Kpeozepze. The results of viscosity measurement are shown in Table 12.

Table 12.

V: t PO HU I OO t U

In table 12 above, the viscosities of the compositions to which adalimumab (100 mg/ml), polysorbate 80 (1 mg/ml) and arginine hydrochloride (AgOoHCl) were added in different concentrations are considered, the viscosity of the solution containing adalimumab ( 100 mg/ml) and polysorbate 80 (1 mg/ml) without AgCHNSiI was 2.11 cP. When AgoNSI was added to the solution in a final concentration from 20 mM to 120 mM, it was confirmed that the viscosities of these compositions decreased in comparison with the composition to which no AgoNSI was added. In the case when AHonNSII was added at a final concentration of 140 mM, the viscosity of the composition was similar to that when AHonNSII was not added. In cases where AgONSI was added at a final concentration of 160 mM or higher, the viscosity-reducing effect was not observed, and the viscosities increased compared to the composition in which AGONSI was not added.

In the case of compositions containing adalimumab (50 mg/ml) and polysorbate 80 (1 mg/ml), the viscosity of the solution without AgoHCI was 1.47 SP, and in cases where Ag9HCI was added at a concentration of 20 mM to 80 mM, the viscosities ranged from 1.42 to 1.45, thus being lower compared to the composition to which AgoNSI was not added. In cases where

AGONSII was added at a final concentration of 100 mM, the viscosity was similar to that when AgOHNSII was not added, and in cases where AgOHNSII was added at a final concentration of 120 mM or higher, the viscosities were increased compared to the case where AgOHNSII was not added.

Accordingly, it was confirmed that in the case of adalimumab solution (100 mg/ml), the viscosity can be reduced by adding Ago9HCII to it at a concentration of 140 mM or less, and in the case of adalimumab solution (50 mg/ml), in viscosity can be reduced by adding to them

AGONSII in a concentration of 100 mM or less. "Example 9"

Experiment 1 confirming the effect of buffers and salts in adalimumab formulations

In order to determine the effect of buffers and salts on the stability of adalimumab, samples of compositions without buffers and salts were obtained, and samples of compositions in which a buffer or salt was added to the compositions obtained above, and these compositions were stored at 40 "C for 2 weeks and 1 month The stability of these samples was compared using 5E-HPLC and the pH of each sample was measured.

Coo)

Table 13 of the stump of

Additional | Initial time at 407 40 "C for 1

Compositions-. . . tion of Mo. Composition buffer and within 2 weeks of the month

No buffer

Adalimumab | Sodium (100 mg/ml), | phosphate/

RBZV8O Sodium

A-2 (1 mg/ml), citrate 0.3810,3910,7610,9112511,410921|227| 319 rno5g (buffer composition

Nitiga)"

After storage After storage at

Compositions - Additional | Initial time at 407 40 "C for 1 st Composition | buffer and within 2 weeks of the month mo. salt Vsyo- Vsyo- leotiye ezg vi oj|oe tia: 2oo ovv| in 55.

Ammonia

A-4 sulfate 0.37 | 0.41 10.78 0.50 | 1.42 | 1.92 2.54 | 3.24 (100 mM)

sodium

A-5 sulfate 0.3910.4110801|0.5411.41/|1.510.751|2.491| 3.24 (100 mM)

No buffer

Sodium phosphate/

sodium

A-7 citrate 0.381 0.41 10.79 | 0.43 | 1.27 | 1.69 2.20 | 2.86

Adalimumab | (buffer (50 mg/ml), composition

RBVO (1 Nitiga)" 52 MM)

Ammonia

A-9 sulfate 0.39|0.41108010,zh8/|1.4511.831 0531/2581 310 (100 mM)

sodium

A-10 sulfate 0.39|0.4410831040|145118510551/|2.511| 3.05 100 mM "Nitiga buffer composition: sodium dihydrogen phosphate dihydrate (0.86 mg/ml), sodium hydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0.3 mg/ml), citric acid monohydrate (1.3 mg/ml)

Table 13 shows the results of ZE-HPLC analysis of the composition of samples at the initial time of each sample, after storage at 40 "C for 2 weeks and after storage at 40 "C for 1 month. Formulation A-1 was prepared to contain adalimumab (100 mg/ml) and

RBZ8O (1 mg/ml), and compositions A-2-A-5 were obtained as containing buffer or salt in composition A-1. In addition, compositions A-6-A-10 were obtained by adjusting the concentration of adalimumab in compositions A-1-A-5 to 50 mg/ml. Overview of the results of 5E-HPLC analysis of each sample, when comparing formulation A-1 without buffer and formulation A-2 without buffer (or comparing formulations A-6 and A-7 with different concentrations of adalimumab), it was found that the formulation without buffer had less increase of NMMU and I MMU, thus confirming that these compositions are more stable.

In addition, when compositions A-1 and A-b6 were compared with compositions containing salt, which includes Masi, ammonium sulfate, sodium sulfate, etc., it was found that compositions without salt had less increase in NMMU and I MUM compared to compositions with salt Accordingly, it was confirmed that in terms of stability, adalimumab compositions that do not contain salts are preferred.

In addition, all storage samples had a constant pH of 5.2 before storage, 2 weeks after storage, and 1 month after storage. Accordingly, it has been confirmed that adalimumab at a concentration of 50 mg/ml or higher has a sufficient intrinsic buffering effect, thus, without requiring the use of a buffer, and without using a buffer, compositions with improved stability can be formulated. "Example 10"

Experiment 2 confirming the effect of buffers and salts in adalimumab compositions

In order to determine the effect of buffers and salts on the stability of adalimumab, samples of compositions consisting of adalimumab (100 mg/ml or 50 mg/ml), stabilizer (sucrose (55 mg/ml) or glycine (160 mM)) were obtained. arginine hydrochloride (AgoNSII: 50 mM), methionine (5

MM), and polysorbate 80 (1 mg/ml), and samples of compositions in which buffer or salt was added to the above compositions. For the purpose of comparison, samples were obtained with the Nitig? composition containing adalimumab (100 mg/ml) or adalimumab (50 mg/ml), and each composition was filled into a glass syringe in the amount of 0.4 ml per syringe, stored at 55 "С for one week, and the stability was compared using 5E-HPLC, and the pH was measured. The details of each formulation and the monomer content before and after storage at 55°C for 1 week are shown in Table 14 below.

Table 14

Before storage After storage

Composition Additional buffer and salt at 5570 at 55 "C for 1 ry for 1 week week

Adalimumab | (77777777-177711177771/ 11117988. | 2 YuyuYushRF8 (100 mg/ml), | Sodium chloride (mass) (100 mM) Sucrose (55 mg/ml), AGONSI | Magnesium chloride (MaSig) (100 mM) (50 mM) , Me! (5 | Calcium chloride (Sasig) (100 mM) wt»

Adalmumab | (77777777-11777777777777117 11171988. | 2 yushC9B2 (100 mg/ml), SIu (160 mm),

AgHCI (50 mM), Magnesium chloride (100 mM) 77777 17711989 | 2 shCG948

ROVOM, i mg/ml), | Sodium phosphate/Sodium citrate pH 52 (Nytigaeu buffer composition

Adalimumab (50 mg/ml), sucrose (55 mg/ml), Ag9NSI

Mu Re (5

MM), Sodium phosphate/Sodium citrate mg/ml), pH 5.2 (Nytigaeu buffer composition

Adalimumab (50% 77777711 1111990 |

MM), Me! (5 mM ZON , mg/ml), ra », Sodium phosphate/Sodium citrate x Composition Nitigaye: sodium dihydrogen phosphate dihydrate (0.86 mg/ml), sodium hydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0 .3 mg/ml), citric acid monohydrate (1.3 mg/ml), mannitol (12 mg/ml), sodium chloride (6.16 mg/ml), PZ8O (1 mg/ml). "x Buffer composition Nitigaf): Sodium dihydrogen phosphate dihydrate (0.86 mg/ml), Sodium hydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0.3 mg/ml), citric acid monohydrate (1.3 mg /ml).

First, all storage samples had a constant pH of 5.2 before and after storage at 557 for 1 week. Accordingly, it was confirmed that adalimumab at a concentration of 50 mg/ml or higher has a sufficient internal buffering effect in the compositions and similar compositions in Table 14 above.

As shown in Table 14 above, the monomer contents of the samples were similar to each other, ranging from 98.8 95 to 99.0 95. After storage at 55 "C for 1 week, the monomer contents were shown to be different in depending on the compositions. Monomer content in sample A-11 with the composition of adalimumab (100 mg/ml), sucrose (55 mg/ml), AgoNSI (50 mM), methionine (5 mM), and polysorbate 80 (1 mg/ml) , was 94.8 95 after storage. However, in the cases of samples A-12-A-16, in which the following salts were used: sodium chloride, ammonium sulfate, magnesium chloride, and calcium chloride, or sodium citrate buffer, the monomer content after storage was in ranged from 94.1 95 to 94.7 95, which was lower compared to the sample formulated in which no salt and buffer were used.In the case of sample A-17, in which Su (160 mM) was used as a stabilizer instead of sucrose, monomer content after storage was 95.2 95; however, in the cases of samples

A-18-A-23, in which salts and buffers were used, the monomer content after storage was in the range from 94.0 95 to 94.9 95, thus being lower compared to the sample with a composition in which salt and buffer did not use Even when the formulation was prepared with a reduction of adalimumab to 50 mg/ml, when sucrose was used as a stabilizer without the use of salt and buffer (i.e., sample A-25), the monomer content after storage was 95.2 95, and when glycine was used (Su) as a stabilizer without the use of salt and buffer (i.e., sample A-32), the monomer content after storage was 95.6 95. However, when additional salt/buffer was used in each composition, in the cases of compositions (samples A-26- A-31), when sucrose was in the composition, the monomer content after storage was in the range from 93.5 95 to 95.0 95, and in the cases of compositions (samples A-33-A-38), when Su was in the composition, the monomer content after storage ranged from 93.6 95 to 95.4 95 , thus showing that the monomer content in the compositions in which additional salt buffer was used was lower compared to the composition in which salt and buffer were not used. Accordingly, it was confirmed that when the additional salt and buffer were not used in compositions comprising adalimumab, arginine, a stabilizer, and surfactants, the stability of adalimumab could be improved, and that it was possible to obtain a composition with improved stability by using the internal buffers the effect of adalimumab itself, without the use of an additional buffer.

However, in the comparison of samples A-24 and A-39 with the Nitiga? composition, which has the same content of adalimumab, the monomer content in the compositions, which included salt and buffer, after storage at 55 "С for 1 week, were more compared to samples with a composition

Nitiga? after storage at 55 "C for 1 week.

Accordingly, from the standpoint of stability, it is preferable that in the compositions of adalimumab, which include arginine, surfactants and stabilizers, no additional salt and buffer were used; however, formulations containing arginine, surfactants, and stabilizers have been shown to be more stable than commercial Nytigages formulations, whether or not these formulations contain additional salt and buffer. "Example 11"

An experiment to confirm the stabilizing effect of polyols in adalimumab compositions

To compare the stabilizing effect of polyols used to increase the stability of adalimumab in solution, adalimumab solution (112 mg/ml adalimumab) and a composition containing adalimumab at a concentration of 112 mg/ml and each type of polyol at a concentration of 42 mg/ml were prepared as follows . The content of NMUU, HMUU and monomers was analyzed using 5E-HPLC after 5 cycles and 10 cycles of repeating the freezing/thawing process at -70 "C and 5 "C, respectively.

Table 15

Cho Cho Cho Cho Cho Cho Cho Cho Cho

Manit (42 mu/ml 0391041 080 /051Щ|048| 099 |06510,936| 01

Table 15 shows the composition of the samples and the results of 5E-HPLC analysis of the samples according to the sample selection points in the stability study. Regarding the above results, when each sample is subjected to the repeated freeze/thaw process, the increase of NMUU and GMUM in the formulations to which mannitol, sucrose, or trehalose were added tended to decrease compared to the formulation to which no polyol was added, thereby confirming the presence stabilizing effect of polyols. Comparing the stabilizing effect of each polyol depending on its type, when sucrose or trehalose was added, the content of NMUM and HMUU was similar to the samples before the freeze/thaw process, and in addition, the degree of purity was shown to be similar to the degree of purity before by undergoing the freeze/thaw process even after they had undergone 10 cycles of the freeze/thaw process, thereby confirming the significant stabilizing effect of polyols.

In contrast, in the case of the sample containing mannitol, when the sample was subjected to a repeated freeze/thaw process, it was confirmed that the NMUM content tended to increase and the purity of the sample decreased during the freeze/thaw process.

Accordingly, it was confirmed that sucrose and trehalose have a greater stabilizing effect compared to mannitol. "Example 12"

A freeze/thaw experiment of adalimumab formulations to confirm and compare the stabilizing effects of arginine, methionine, glycine and sucrose.

To confirm the stabilization effects of arginine, methionine, glycine and sucrose during the freeze/thaw process of stock solutions, samples were prepared as shown below.

Each sample in the amount of 1 ml was added to a polycarbonate vial (5 ml), subjected to 5 cycles of the freeze/thaw process during preparation, and at -70 C and 5 "C, respectively, and analyzed using 5E-HPLC. Composition of each sample and results 5E-HPLC for each sample before and after the freeze/thaw process is shown below.

Table 16

Before After 5 cycles

Composition AgaTNSI | sapu Mei |sucrose by freezing/ Freezing/

M) (mM) (mM) (mg/ml) thawing thawing (m 7-1 - 1-1 - |o0z2|o040| 072176 | 028 | 204 ddalimumab 3501-11 - 10961039 o74| 0671031 098 bzomom 10350115 1 - 08341087 0110671 0331 1007 olisorbatvo 3501-1251: 1033 034 | 067052 0.93 0857 (1 mg/ml) 50 |160| 5 | - |034| 096070 | om | 031 | 073 50 |140| 25 | - |0933|054 | 066 | 043 0.33 | 0.75 5 | 0 | 5 | 55 | 034 | 055069 | ohm | 0.29 | 0.70

Samples were obtained to contain adalimumab (130 mg/ml) and polysorbate 80 (1 mg/ml) and to contain an additional stabilizer therein. The impurity content of each sample before the freeze/thaw process was shown to be similar to each other for both NMSU and

MM. After the freeze/thaw process, the I MMU content was shown to be similar in all samples; however, the content of NMMUU has been shown to vary depending on the type and content of additional stabilizer. In the case of a sample to which no additional stabilizer was added, the content

NMMU increased to 1.76 95 after 5 freeze/thaw cycles, while in the case of the sample containing arginine hydrochloride (50 mM), the NMMU content significantly decreased to 0.6795 after 5 freeze/thaw cycles. In the case of the formulation in which methionine was added in addition to arginine hydrochloride (50 mM), no additional reduction in NMU was confirmed when the formulation contained methionine at mM concentration, whereas NMU was further reduced by approximately 0.52 95 when the formulation contained methionine at a concentration of 25 mM. In cases where glycine or sucrose was added in addition to 5 arginine hydrochloride and methionine, it was confirmed that the NMMUU content after 5 freeze/thaw cycles decreased further to be in the range of 0.41 95 to 0.43 95, and both the glycine-containing composition and the sucrose-containing composition showed similar stability. Accordingly, methionine, arginine, glycine, and sucrose were all confirmed to contribute to the stability of adalimumab. In addition, it has been confirmed that similar levels of stability can be achieved when the compositions are prepared by appropriate combinations using a polyol or amino acid as a stabilizer. "Example 13"

Comparative stability experiments among compositions containing glycine, compositions containing sucrose, and commercial compositions of NitigaFeF

To compare the stability of compositions containing glycine, compositions containing sucrose, and commercial Nitigaf compositions, samples were obtained with the content of adalimumab at a concentration of 100 mg/ml or 50 mg/ml, arginine hydrochloride (AgoNSiII) at a concentration of 50 mM, polysorbate 80 ( RBZ80) at a concentration of 1 mg/ml, and methionine at a concentration of 5 mM, and then additional samples were obtained by adding glycine, a combination of glycine and methionine, or sucrose as an additional stabilizer to the above compositions. In addition, samples were obtained to have commercial formulations of Nitigaf and to contain adalimumab at a concentration of 100 mg/ml or 50 mg/ml. Each of the above samples was loaded into 1 ml glass syringes in the amount of 0.4 ml per syringe. After storing each syringe at 40 "C for 4 weeks, the contents were analyzed

NMMIU and GMMU in each sample, using BE-HPLC. Composition of each composition and results

ZE-HPLC is shown in Table 17 below.

Table 17. o After storage at . Before storage at 40 C o

Composition Additional for 4 weeks 407c stabilizer for 4 weeks

Adalimumab (100 | (g - | 028 | 0.39 | 067 | 0.54 | 244 | 299 mg/ml), Agr"NSI (50 | su (120 mM)

MM), RZVO (1 mg/ml), | Su (160 MM) 028 | 042 | 069 | 054 | 237 | 291

Me! (5 mM

Sucrose (55 mg/ml)

Adalimumab (100 mg/ml), Composition

Nitigaye 0.40 0.40 0.80 0.98 3.05 4.03

Adalimumab (50 7 - yuyu / | 027 | 038 | 064 | 035 | 296 | 271 mg/ml), Agr"NSI (50 | siu (120 mM)

MM), RZVO (1 mg/ml), | Su (160 mm)

Me! (5 mM

Sucrose (55 mg/ml)

Adalimumab (50 mg/ml), Composition Nitigaye

Samples of formulations containing arginine hydrochloride, polysorbate 80, methionine, and adalimumab showed similar levels of stability before storage at 40°C for 4 weeks.

Over time, samples with a commercial composition of Niptig? showed a higher content of NMU by approximately 0.1 95 when obtained and in comparison with other samples. Regarding the total content of NMUM and HMUU after storage, all samples containing arginine hydrochloride showed relatively low values: ie, from 2.81 95 to 2.99 95 when adalimumab was contained at a concentration of 100 mg/ml; and from 2.56 95 to 2.71 95 when adalimumab was contained at a concentration of 50 mg/ml. However, in the case of commercial compositions of Nitiga?, the total content of NMUM and I MUM after storage was 4.03 95, when adalimumab was contained in a concentration of 100 mg/ml; and 4.06 95 when adalimumab was contained in a concentration of 50 mg/ml, thus showing higher levels of NMUM and I MUM content compared to samples of compositions containing arginine. Accordingly, it was confirmed that compositions containing combinations of additives described in the examples of the invention; that is, compositions containing arginine and compositions containing a polyol or amino acid as an additional stabilizer are superior to commercial NitigaF compositions in terms of stability. "Example 14"

Comparative experiments on the stability of adalimumab compositions depending on the concentration of adalimumab, sucrose, glycine, leucine, methionine, sodium chloride (mass) and arginine

To compare the stability of adalimumab compositions, samples of different composition were obtained by combining adalimumab, arginine hydrochloride (AgoHCl), sodium chloride (mass), polysorbate 80 (RBZ80), methionine (Me), sucrose, glycine (Su), and leucine (He). In addition, samples containing commercial compositions of NiptigaF and containing adalimumab at a concentration of 100 mg/ml or 50 mg/ml were obtained for comparison purposes. Each composition was loaded into a 1 ml glass syringe in the amount of 0.4 ml, stored at 40 "C for 4 weeks, and the monomer content was analyzed before and after storage using ZE-HPLC. Compositions and results 5E-

HPLC is shown below.

Table 18

Before after

Adalimu- stored- stored mab Agansi| mass |Mei| RBZVO |Sucrose| Sioux | Heine at 402C (mg/ml) (mM) | (mM) KmM))| (mg/ml) | (mg/ml) |(mM))/| (mM) 407 for 4 weeks 4 weeks

AYA4o | 100 | 50 | |51 1 | 55 | | | 9929 | 975

And | lo | 50, | 51 1 | 45 | | | 9933 | 9716

Ah | 100 | 50 | Sh|251 1 | 45 | | | 99530 | 974 43 | 100 | 50 | |51 1 | 45 |201 sh | 9930 | 978 44 | forehead | 50 | | 51 1 | 45 | | 20 | 9930 | 972 | 100 | 50 | |51 1 | 35 | | | 9930 | 97.06 46 | 100 | 50 | Sh|251 1 | 935 | | | 9930 | 974 47 | forehead | 50 | 251 1 | z5 |401 | 99339 | 97.09 48 | 100 | 50 | (251 1 | 35 /|20| 20 | 9929 | 97293

A-49 | 100 | 50 | |5th 1 | 25 | | | 9931 | 9/1

OR | 100 | 50 | |251 1 | 25 | | | 9932 | 973

AB | 100 | 50 | |251 1 | 25 |601 | 9930 | 977

A-52 | 100 | 50 | |251 1 | 25 |401 20 | 9931 | 977

A-B5Z3 | 100 | 50 | |5th 1 | sh | her | 9931 | 9706

A-5A4 | 100 | 50 | |251 1 | ShB | her | 9932 | 973

AB | 100 | 50 | |251 1 | - 1401 | 9930 | 9704

A-Bb | 100 | 50 | 50 | 5 1 | 25 | | | 99.31 | 96,96

ABU | 100 | 50 | 50 |15 1 | 25 | | | 9928 | 9693

A-5B | 100 | 50 | 50 |251 1 | 25 | | | 9927 | 96489

A-5O | 100 | 25 | 60 | 5 1 | 25 | | | 99.30 | 9688

A-vo | 100 | 25 | 60 |35 1 | 25 | | | 9929 | 9700

A-v1 | 100 | 25 | 60 |51 1 | with 5 | | | 9927 | 9685

A-b3 | 50 | 50 | |51 1 | 55 | | | 9930 | 9723

A-b4 | 50 | 50 | |51 1 | 45 | | | 9930 | 97

A-8B5 | 50 | 50 | |251 1 | 45 | | | 9931 | 9728

A-b6 | 50 | 50 | sh /5| 1 | 45 |20| | 9930 | 9730

Before after

Adalimu- stored- stored mab Agansi| mass |Mei| RBVO |Sucrose| SIu | And the reaction at 402С (mg/ml) (mM) | (mM) KmM))| (mg/ml) | (mg/ml) |(mM)/| (mM) 407 for 4 weeks 4 weeks

A-b7 | 50 | 50 Sh |5Y| 1 | 45 | | tho | 9930 | 9732

AB | 50 | 50 Sh |5Y| 1 | with 5 | | | 9930 | 9723

A-b9 | 50 | 50 sh |25| 1 | with 5 | | | 9931 | 9723

A-7O | 50 | 50 shsh |25| 1 | from 5 140| | 9930 | 9727

A-7I | 50 | 50 shshsh |25| 1 | z5 1201 20 | 9930 | 9734 72 | 50 | 50 Shsh(|5Y| 1 | 25 | | | 9931 | 975

A-73 | 50 | 50 sh |25| 1 | 25 | | | 9930 | 9724

A-74 | 50 | 50 sh-|25| 1 | 25 1|60| | 9930 | 9732

A-75 | 50 | 50 |25| 1 | 25 140| 20 | 9931 | 9726

A-7b | 50 | 50 шЩщ |5Й 1 ІІ Х From Her | | 9929 | 9725

A-77 | 50 | 50 shshsh |25| 1 | shR Her | | 9930 | 973

A-78 | 50 | 50 sh |25| 1 | B 140! | 9929 | 9733

A-79 | 50 | 50 sh |25| 1 | (ХГ /1t120| h | 9931 | 978

A-8O | 50 | 50 50 |5| 1 | 25 | | | 9929 | 9706

AB | 50 | 50 50 |15| 1 | 25 | | | 9930 | 96.90 82 | 50 | 50 50 |25| 1 | 25 | | | 9927 | 9702 83 | 50 | 25 60 |5| 1 | 25 | | | 9930 | 9681

A-84 | 50 | 25 60 |935| 1 | 25 | | | 9930 | 9700

A-85 | 50 | 25 | 60 |5| 1 | with 5 | | | 9929 | 96.92 " Commercial compositions of Nitigafe: Sodium dihydrogen phosphate dihydrate (0.86 mg/ml), sodium hydrogen phosphate dihydrate (1.53 mg/ml), sodium citrate (0.3 mg/ml), citric acid monohydrate (1.3 mg/ml), mannitol (12 mg/ml), sodium chloride (6.16 mg/ml), RBZ80 (1 mg/ml)

The monomer content of all samples before storage at 40 "С were similar to each other, in the range from 99.27 95 to 99.33 o, regardless of the compositions. Regarding the monomer content in the samples (with

A-40 to A-61) containing adalimumab (100 mg/ml) after storage at 40 "C for 4 weeks, in the case of samples (from A-40 to A-55) that did not contain sodium chloride, the content of monomers was in the range from 97.04 95 to 97.23 965, while in the case of samples containing sodium chloride (from A-5b6 to A-61i with the exception of A-62, which is a composition of NitigafF), the content of monomers was in range from 96.85 95 to 97.00 95, thus showing a slightly lower monomer content compared to those compositions that did not contain sodium chloride However, the difference in monomer content between these samples depending on the content of sodium chloride, sucrose , methionine, glycine and leucine after storage at 40 C for 4 weeks is shown to be relatively insignificant, and the monomer content in compositions (from A-40 to A-61) is shown to be higher than 95.64 95, monomer content in A-62, which is a composition of Nitigat containing adalimumab in the same concentration, by at least 1 95.

In the case when adalimumab was contained in a concentration of 50 mg/ml, the results of the analysis were the same as in the compositions in which adalimumab was contained in a concentration of 100 mg/ml.

Regarding the monomer content of samples containing adalimumab (50 mg/ml) (from A-63 to A-85), after storage at 40 °C for 4 weeks, in the case of samples from A-63 to A-79, in which did not contain sodium chloride, the monomer content was in the range from 97.15 95 to 97.38 905, while in the case of samples containing sodium chloride (from A-80 to A-85, with the exception of A-86b, which is a composition

Nitigaf), the monomer content was in the range from 96.81 95 to 97.06 95, thus showing a slightly reduced monomer content compared to those compositions that did not contain sodium chloride. However, the difference in the monomer content between these samples depending on the content of sodium chloride, sucrose, methionine, glycine and leucine after storage at 40 "C for 4 weeks, as shown, is relatively insignificant, and the monomer content in the compositions (from A-63 according to A-85), as shown, is higher than 95.47 95, the content of monomer A-86, which is a composition of NitigafF containing adalimumab in the same concentration, by at least 1 95.

Accordingly, it was confirmed that combinations of additives and their compositions described in the examples of the invention, i.e. compositions containing arginine, are superior to commercial compositions

Nitigat regarding stability.

From the foregoing, one skilled in the art to which the invention relates will be able to understand that the invention may be implemented in other specific forms without modifying the technical concepts or essential characteristics of the invention. Accordingly, the illustrative embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the invention.

Rather, the invention is intended to encompass not only the illustrative embodiments, but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the invention as defined in the appended claims.

Claims (22)

FORMULA OF THE INVENTION 1. A liquid composition of an antibody against TME-os, which contains an antibody against TME-o, a stabilizer, a surfactant, an antioxidant and arginine, where the liquid composition does not contain an additional buffer and where the antibody is against TME-o; is adalimumab. 2. The liquid composition according to claim 1, in which the stabilizer is a polyol, an amino acid other than arginine, or a combination thereof. 3. The liquid composition according to claim 2, in which the polyol is selected from sucrose, trehalose, PEG, and combinations thereof. 4. The liquid composition according to claim 2, in which the amino acid is selected from glycine, leucine, isoleucine, phenylalanine, proline, and combinations thereof. 5. Liquid composition according to claim 1, in which the stabilizer is selected from the group consisting of: () sucrose or trehalose; (iii) PEG having an average molecular weight of 200 to 600 or PEG having an average molecular weight of 1000 to 8000; (iii) glycine or leucine; and (im) combinations of at least two of (1)-(iii). Zo 6. Liquid composition according to claim 2, in which the polyol is present in a concentration of 0.1 to 100 mg/ml. 7. The liquid composition according to claim 2, in which the amino acid other than arginine is present in a concentration of 1 to 300 mm. 8. The liquid composition according to claim 1, in which the surfactant is a nonionic surfactant. 9. Liquid composition according to claim 1, in which the surfactant is a polysorbate or a poloxamer. 10. Liquid composition according to claim 9, in which the surfactant is polysorbate 80, polysorbate 20 or poloxamer 188. 11. Liquid composition according to claim 1, in which the surfactant is present in a concentration of 0.1 to 5 mg/ml. 12. Liquid composition according to claim 1, in which arginine is in the form of a salt. 13. The liquid composition according to claim 12, in which the arginine is in the form of arginine hydrochloride. 14. Liquid composition according to claim 1, in which arginine is present in a concentration of 0.1 to 200 mM. 15. The liquid composition according to claim 1, in which the antibody is against TME-o; is present in a concentration from 1 to 250 mg/ml. 16. The liquid composition according to claim 1, in which the antibody is against TME-o; is present in a concentration of 50 to 200 mg/ml. 17. Liquid composition according to claim 1, in which the antioxidant is methionine. 18. Liquid composition according to claim 17, in which methionine is present in a concentration of 1 to 50 mM. 19. A liquid composition according to any one of claims 1-18, which has a pH of 4 to 6. 20. Liquid composition according to claim 1, which contains: an antibody against TME-o; in a concentration from 1 to 250 mg/ml; polyol in a concentration from 0.1 to 100 mg/ml; surfactant in a concentration of 0.1 to 5 mg/ml; and arginine in a concentration from 0.1 to 200 mM. 21. Liquid composition according to claim 1, which contains: an antibody against TME-o; in a concentration from 1 to 250 mg/ml; an amino acid other than arginine in a concentration of 1 to 300 mM; surfactant in a concentration of 0.1 to 5 mg/ml; and arginine in a concentration from 0.1 to 200 mM. 22. The method of obtaining a liquid composition according to claim 1, which consists in mixing an antibody against TME-o, a stabilizer, a surfactant, an antioxidant and arginine, where the antibody against TME-o is adalimumab. Whiteness SP) Bu ZY - y o ХК 3 58 as 15 y y ; and; 1st: thank you with ZA Za ZY fedry tyr snya V y dr m otv zhyut ti YOU M vk as hom vu dr me dru shiny she she she she shen more sh- -O zi Moh still VUM M a SE yah to O I still ha I OH still KK o KN KV I W U Z KOH OV XX U OH U I ZO KE KE to U VH OK SO Ko I M scha Me Me EO PE M PE I M MU HO SO still still M Mo he and I VE SE OO JAZ Z o 5 same S that U U 5 U U 5 5 shi she she she she she she she she she she she she she she she she Ko OK SU ME Ky OH S sht po penyazzhyuyu oi nevy seni oo M tychuzhynni oyu penyu v syu VO nya shk huya ey suhu a slu ei kakhkyuni sha sokhkuk VK nkyu X x KU Z Z z U Ka Ku KU Z Z y y Z х г я хЭ х Ж 5 щ З с У, и хм Ko е - Х 5 5 g Е и х - Ж 5 ГУ х х KE ш Х Ух EE х M m ж y Х Й KO КУ г г. 1 Concentration of particles (we) pr um et ok t Yani telyat MV FACTORIES donna nnion VV don y Kydynia Sary i SE nn NN B nya VV Kompozitsov 45 i IN oy"; ak A To pkchnh d Khompolyaviv 45 ! ж ХВ mrosnnkeyutnisntnlyuyunnnmnkh tk yunyanuki children ktkltzhh dec zh tk ste nki khnih "x AGAIN kny UK sia N EK V i Yad p nn sy NN NK U ido passage song passage di passage after passage Ii zerezhnasvzs | ожежввосю ой зережнаесс В through the new os : ! ! ! ! and | . E and PLACEVA SAMPLE WHICH ADALIMUTAV INVESTS: fig. 2