Formulations for anti-FCRN antibodies

Abstract

In one embodiment of the present invention, there is provided a pharmaceutical formulation having a pH of 4.0 to 8.0, comprising (a) an HL161BKN antibody or a fragment thereof, (b) at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine, and salts thereof, (c) a buffer system selected from citrate or histidine, and (d) a surfactant. The HL161BKN present in the formulation has improved stability and is non-toxic, thus having high potential for industrial applications.

Description

Technical Field

[0001] The present invention relates to a formulation optimized for HL161BKN, an anti-FcRn antibody.

Background Art

[0002] The causes of autoimmune diseases have been studied for a long time from genetic, environmental, and immunological perspectives, but the exact causes of the diseases are still unknown. Many recent studies have revealed that many autoimmune diseases are caused by IgG-type autoantibodies. In fact, in the research on the diagnosis and treatment of autoimmune diseases, the relationship between the presence or absence of disease-specific autoantibodies and the therapeutic effect due to their reduction has been widely studied.

[0003] As a therapeutic agent for such autoimmune diseases, systemic injection of high-dose steroids is the first choice drug, and high-dose IVIG (intravenous immunoglobulin) administration or plasmapheresis is applied when the symptoms are severe and difficult to control with steroids. High-dose steroids may have a weak effect or serious side effects when used repeatedly. In the case of IVIG and plasmapheresis, since the treatment cost is high and there are various side effects and infection risks, the development of therapeutic drugs in this treatment field is urgently needed.

[0004] On the other hand, recently, therapeutic drugs for autoimmune diseases using FcRn antibodies have been studied (Korean Patent Publication No. 10-2014-0147606). It is a drug having a new strategy of inhibiting FcRn (Neonatal Fc Receptor), which the antibody is involved in the recycling of IgG, enhancing the catabolic action of IgG in the body, thereby reducing the level of autoantibodies and treating the disease. This anti-FcRn antibody is expected as a product that can solve the problems of existing therapeutic drugs.

[0005] However, in order to apply this antibody to severe autoimmune diseases such as pemphigus vulgaris, neuromyelitis optica, and myasthenia gravis, which are caused by the production of autoantibodies against autoantigens in the body, there is a need for formulations of this antibody optimized for both the antibody itself and the method of administration. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Korean Patent No. 10-2014-0147606, Specification A [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Disclosure of the invention Technical problems As a result of research aimed at developing optimized formulations for anti-FcRn antibodies to treat severe autoimmune diseases, the inventors have developed optimized buffers and formulations for the anti-FcRn antibody HL161BKN. [Means for solving the problem]

[0008] Solution to the problem To achieve the above objective, in one aspect of the present invention, a pharmaceutical formulation is provided comprising (a) an anti-FcRn antibody or a fragment thereof, (b) at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine and salts thereof, (c) a buffer system selected from citrate or histidine, and (d) a surfactant. [Effects of the Invention]

[0009] A pharmaceutical formulation according to the present invention having a pH of 4.0 to 8.0, comprising (a) an anti-FcRn antibody or a fragment thereof, (b) at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine and salts thereof, (c) a buffer system selected from citric acid or histidine, and (d) a surfactant, is an optimized pharmaceutical composition for HL161BKN, and it has been confirmed that the stability of HL161BKN in the formulation is improved. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 demonstrates the results of analyzing the thermal stability of HL161BKN using DSC (Differential Scanning Calorimetry). [Figure 2] Figure 2 demonstrates the results of the 4-week accelerated stability test of HL161BKN under sodium citrate-phosphate buffer conditions of pH 5.0, 6.0, 7.0, and 8.0. [Figure 3] Figure 3 is a schematic diagram of the process for considering the formulation of HL161BKN. [Figure 4] Figure 4 demonstrates the results obtained by examining the changes in aggregates and fragments of HL161BKN (210 mg / mL) due to various excipients in the excipient tests. [Figure 5] Figure 5 demonstrates the results of analyzing the charge variant patterns of HL161BKN (210 mg / mL) by CEX-HPLC under different excipient conditions. [Figure 6] Figure 6 demonstrates the results of analysis of variance (ANOVA) of the change in HL161BKN aggregates depending on the combination of excipients. [Figure 7] Figure 7 demonstrates the results of examining the changes in aggregates and fragments of HL161BKN (210 mg / mL) depending on the combination of excipients. [Figure 8]Figure 8 demonstrates the results of an analysis using Design of Experiment (DOE) to determine the changes in HL161BKN aggregates and fragments depending on the combination of excipients. [Figure 9] Figure 9 demonstrates the results of CEX-HPLC analysis of the charge variant patterns of HL161BKN based on excipient combinations. [Figure 10] Figure 10 illustrates the results of further excipient testing, examining the changes in aggregates and fragments of HL161BKN (210 mg / mL). [Figure 11] Figure 11 demonstrates the results of analyzing the charge variant patterns under different excipient conditions in further excipient testing of HL161BKN using CEX-HPLC. [Figure 12] Figure 12 demonstrates the results of confirming the effect of HL161BKN (210 mg / mL) on stirring stress with and without PSB20. [Figure 13] Figure 13 demonstrates the results of the stirring test, showing the change in monomers of HL161BKN with and without PSB20. [Figure 14] Figure 14 demonstrates the results obtained by examining the changes in aggregates and fragments of HL161BKN (210 mg / mL) in the viscosity-reducing excipient test. [Figure 15] Figure 15 demonstrates the results obtained by observing changes in aggregates and fragments of HL161BKN (210 mg / mL) during the first excipient screening. [Figure 16] Figure 16 demonstrates the results obtained by analyzing the charge variant pattern of HL161BKN according to the first excipient screening conditions using CEX-HPLC. [Figure 17] Figure 17 demonstrates the results obtained by observing changes in aggregates and fragments of HL161BKN (210 mg / mL) during the second excipient screening. [Figure 18]Figure 18 demonstrates the results obtained by analyzing the charge variant pattern of HL161BKN under the second excipient condition using CEX-HPLC. [Figure 19] Figure 19 shows the results obtained by confirming the change in the number of insoluble microparticles of HL161BKN in Figure 19. [Figure 20] Figure 20 shows the results obtained by confirming the viscosity according to the concentration of HL161BKN in the HL161BKN formulation. Here, when the concentration of HL161BKN was 170 mg / mL, the viscosity was confirmed to be 10 cP.

[0011] Detailed Description of Implementing the Invention In one aspect of the present invention, there is provided a pharmaceutical formulation having a pH of 4.0 to 8.0, comprising (a) an anti-FcRn antibody or a fragment thereof, (b) at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine, and their salts, (c) a buffer system selected from citrate or histidine, and (d) a surfactant.

[0012] Furthermore, the pharmaceutical formulation may further contain methionine. Additionally, the pharmaceutical formulation may further contain saccharides such as sucrose and trehalose.

[0013] The buffer system refers to a buffer solution that is resistant to changes in pH by acid-base counterparts.

[0014] As used herein, "histidine buffer" refers to a buffer solution containing histidine ions. Here, the specific form of the histidine buffer may be any one selected from the group consisting of histidine chloride buffer, histidine acetate buffer, histidine phosphate buffer, and histidine sulfate buffer, but is not limited thereto.

[0015] As used herein, "citric acid buffer" refers to a buffer containing citrate ions. Here, a specific embodiment of the citrate buffer may be, but is not limited to, any one selected from the group consisting of sodium citrate buffer, potassium citrate buffer, calcium citrate buffer, and magnesium citrate buffer.

[0016] As used herein, the term “surfactant” refers to a pharmaceutically acceptable excipient used to protect protein formulations from mechanical stress such as agitation and shearing. Specific embodiments of surfactants include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer, triton, sodium dodecyl sulfate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, laurylamidopropyl betaine, The surfactant may be any one selected from the group consisting of cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitoylamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitoylamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methylcocasyl, sodium methyloleyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol. Here, the surfactant is preferably polysorbate 20.

[0017] Furthermore, the pharmaceutical formulation may be an aqueous formulation, preferably an injectable liquid formulation.

[0018] Here, the anti-FcRn antibody may be HL161BKN.

[0019] HL161BKN includes a heavy chain variable region comprising H-CDR1 having the amino acid of SEQ ID NO: 5, H-CDR2 having the amino acid of SEQ ID NO: 6, and H-CDR3 having the amino acid of SEQ ID NO: 7, and a light chain variable region comprising L-CDR1 having the amino acid of SEQ ID NO: 8, L-CDR2 having the amino acid of SEQ ID NO: 9, and L-CDR3 having the amino acid of SEQ ID NO: 10.

[0020] Furthermore, HL161BKN may include the heavy and light chain regions shown in Table 1 below. Additionally, the heavy and light chains may be encoded by the nucleic acids shown in Table 2. [Table 1]

[0021] The glycosylation sites of the antibody are as follows: Asn301, N-glycan (G0F, G1F, G0-GlcNac, Man5). [Table 2]

[0022] Here, the pharmaceutical preparation may have a viscosity of 20 cP or less. Specifically, the pharmaceutical preparation may have a viscosity of 1 cP to 20 cP, and may have a viscosity of 10 cP to 20 cP, and may have a viscosity of about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, about 19 cP, or about 20 cP.

[0023] Furthermore, the pharmaceutical formulation may have an osmotic pressure of 250 mOs / kg to 500 mOs / kg. Specifically, the pharmaceutical formulation may have an osmotic pressure of 300 mOs / kg to 450 mOs / kg or 350 mOs / kg to 400 mOs / kg. Furthermore, the pharmaceutical formulation may have an osmotic pressure of approximately 250 mOs / kg, approximately 260 mOs / kg, approximately 270 mOs / kg, approximately 280 mOs / kg, approximately 290 mOs / kg, approximately 300 mOs / kg, approximately 310 mOs / kg, approximately 320 mOs / kg, approximately 330 mOs / kg, approximately 340 mOs / kg, approximately 350 mOs / kg, approximately 360 mOs / kg, approximately 370 mOs / kg, and approximately 3 It may have an osmotic pressure of 80 mOs / kg, approximately 390 mOs / kg, approximately 400 mOs / kg, approximately 410 mOs / kg, approximately 420 mOs / kg, approximately 430 mOs / kg, approximately 440 mOs / kg, approximately 450 mOs / kg, approximately 460 mOs / kg, approximately 470 mOs / kg, approximately 480 mOs / kg, approximately 490 mOs / kg, or approximately 500 mOs / kg.

[0024] Furthermore, the pharmaceutical formulation may contain HL161BKN at concentrations ranging from 50 mg / mL to 250 mg / mL. Specifically, the pharmaceutical formulation may contain HL161BKN at concentrations ranging from 60 mg / mL to 250 mg / mL, 70 mg / mL to 250 mg / mL, 80 mg / mL to 250 mg / mL, 90 mg / mL to 250 mg / mL, or 100 mg / mL to 250 mg / mL. In addition, the pharmaceutical formulation may contain HL161BKN at concentrations ranging from 120 mg / mL to 230 mg / mL, 150 mg / mL to 220 mg / mL, or 180 mg / mL to 210 mg / mL. Furthermore, the pharmaceutical preparation may contain HL161BKN at concentrations of approximately 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, or 250 mg / mL.

[0025] Furthermore, the pharmaceutical formulation may have a pH of 4.0 to 8.0. Specifically, the pharmaceutical formulation may have a pH of 4.0 to 7.0. Preferably, the pharmaceutical formulation may have a pH of 5.0 to 6.0. Moreover, the pharmaceutical formulation may have a pH of approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, approximately 5.7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, or approximately 7.0.

[0026] Furthermore, additives may be included at concentrations ranging from 10 mM to 400 mM. Here, mannitol, sorbitol, arginine, histidine, or glycine may be used individually or in combination of two or more. Specifically, each additive may be included at concentrations ranging from 10 mM to 400 mM, 20 mM to 300 mM, 50 mM to 250 mM, or 100 mM to 150 mM. Specifically, the additives may be included at concentrations of approximately 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, or 300 mM.

[0027] Furthermore, two additives may be used in combination. In one embodiment, the additives may include mannitol and sorbitol. In one embodiment, the additives may include mannitol and arginine. In one embodiment, the additives may include mannitol and histidine. In one embodiment, the additives may include mannitol and glycine. In one embodiment, the additives may include sorbitol and arginine. In one embodiment, the additives may include sorbitol and histidine. In one embodiment, the additives may include sorbitol and glycine. In one embodiment, the additives may include arginine and histidine. In one embodiment, the additives may include arginine and glycine. In one embodiment, the additives may include histidine and glycine. Here, each additive may be included in the pharmaceutical formulation at the concentrations described above.

[0028] One embodiment of the pharmaceutical formulation may be a pharmaceutical formulation having a pH of 5.0 to 6.0, comprising (a) 100 mg / mL to 250 mg / mL of anti-FcRn antibody, (b) 50 to 250 mM of L-arginine or its hydrochloride, (c) 50 to 250 mM of L-histidine, and (d) 0.01 to 0.05% polysorbate 20.

[0029] Here, the anti-FcRn antibody is as described above. Furthermore, the pharmaceutical preparation may be an aqueous preparation, or an injectable liquid preparation. Moreover, the pharmaceutical preparation may be administered subcutaneously.

[0030] Furthermore, the formulation was confirmed to be highly stable under accelerated conditions. Specifically, after a 6-month test under accelerated conditions (25°C, 60% relative humidity), the aggregate and fragment content may be approximately 10% or less. Moreover, the aggregate and fragment content under the above conditions may be approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5.5% or less, or approximately 5.0% or less.

[0031] Furthermore, it was confirmed that the formulation remains stable even under long-term storage conditions. Specifically, the content of aggregates and fragments under conditions of 5°C for 36 months may be approximately 10% or less. In addition, the content of aggregates and fragments under the above conditions may be approximately 9% or less, approximately 8% or less, approximately 7% or less, approximately 6% or less, approximately 5% or less, approximately 4% or less, approximately 3% or less, approximately 2% or less, approximately 1.8% or less, approximately 1.5% or less, or approximately 1.2% or less.

[0032] Furthermore, the pharmaceutical formulation may be used to treat autoimmune diseases. Here, the autoimmune disease may be any one selected from the group consisting of myasthenia gravis (MG), thyroid eye disease (TED), hyperthermic autoimmune hemolytic anemia (WAIHA), neuromyelitis optica (NMO), immune thrombocytopenic purpura (ITP), pemphigus vulgaris (PV), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis (LN), and membranous nephropathy (MN).

[0033] Screening of optimized formulations for HL161BKN HL161BKN was prepared at a concentration of approximately 210 mg / mL under each condition, stored for 4 weeks under accelerated conditions at 40°C, and analyzed for concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, osmotic pressure, and insoluble microparticles (MFI) to evaluate the stability of the sample and its suitability for subcutaneous administration.

[0034] First, screening tests were conducted on 11 excipients frequently used in existing antibody products. As a result, L-histidine, L-arginine hydrochloride, L-glycine, D-sorbitol, and D-mannitol were confirmed to have anti-aggregation effects.

[0035] The synergistic effect was tested with five selected excipient combinations. The results showed that L-arginine hydrochloride reduced aggregate formation to a statistically significant level (p<0.01).

[0036] Based on a combination of comparative results regarding the effectiveness of different excipients against aggregation and fragmentation, L-histidine and D-mannitol were selected as excipients. Further screening of excipients led to the selection of L-methionine, which exhibits an inhibitory effect on aggregation formation. It was also confirmed that histidine can be used as a basal buffer. Furthermore, high-purity 0.02% polysorbate 20 was selected to suppress aggregation formation caused by stirring stress that may occur during product storage and transportation.

[0037] Next, a first concentration screening test was performed with the selected histidine basal buffer and the L-arginine hydrochloride, D-mannitol, and L-methionine excipients. The PBS20 concentration was fixed at 0.02%. The results showed high stability under the condition of 50 mM histidine basal buffer without excipients. Furthermore, there was no significant difference in the increase of aggregates and fragments under the excipient conditions, although aggregate formation decreased as the concentration of L-arginine hydrochloride increased.

[0038] On the other hand, it was confirmed that there was almost no difference depending on the presence or absence of D-mannitol and the L-methionine concentration. Therefore, L-arginine hydrochloride and 0.02% PSB20 were selected as HL161BKN preparations in L-histidine basal buffer.

[0039] Finally, a second concentration screening was performed using two batches of HL161BKN to select the optimal concentrations of L-histidine and L-arginine hydrochloride. The results showed no significant difference in the increase in aggregates and fragments under different excipient concentration conditions, and the viscosity and osmotic pressure measurements under all conditions were confirmed to be suitable for subcutaneous administration, being less than 20 cP and 250 to 500 mOsmol / kg.

[0040] Furthermore, analysis of insoluble microparticles using microflow imaging (MFI) was performed on each sample condition, and the condition with the least increase in the number of insoluble microparticles was identified; stability and suitability for subcutaneous injection (SC) administration were evaluated through purity (aggregates and fragments), viscosity, and osmotic pressure tests to select the final formulation.

[0041] In conclusion, approximately 100 mM L-histidine, approximately 100 mM L-arginine hydrochloride, and approximately 0.02% polysorbate 20 (pH 6.0) were selected as formulations for HL161BKN for nonclinical and Phase 1 clinical trials.

[0042] The present invention will be described in more detail below through examples. It will be apparent to those skilled in the art that these examples are for demonstrating the present invention only and should not be construed as limiting the scope of the present invention.

[0043] I. Preparation of HL161BKN Preparation Example 1. Preparation of gene constructs for preparing HL161 antibodies and vectors containing them. To prepare HL161BKN, a polynucleotide having the nucleic acid sequence of SEQ ID NO: 3, which encodes a heavy chain containing the amino acid sequence of SEQ ID NO: 1, was loaded into the pCHO 1.0 vector (Life Technologies). Furthermore, a polynucleotide having the nucleic acid sequence of SEQ ID NO: 4, which encodes a light chain containing the amino acid sequence of SEQ ID NO: 2, was loaded into the pCHO 1.0 vector.

[0044] Preparation Example 2. Preparation of HL161BKN cell line and production of HL161BKN CHO-S cells were transformed using the expression vector prepared in Preparation Example 1, and then subjected to a selection process with methotrexate and puromycin to prepare the final production cell line. The prepared cell line was prepared, stored as a cell bank, and used for HL161BKN production. Antibody production was carried out in a bioreactor containing culture medium (Dynamis medium + 8 mM L-glutamine) by adding supplemental medium (EFB+) every two days, and the supernatant was collected after approximately 15 days of culture. Subsequently, Protein A column chromatography and virus inactivation were performed at low pH, followed by anion exchange chromatography (AEX) and cation exchange chromatography (CEX). After concentration and buffer exchange (ultrafiltration / diafiltration), it was purified by sterile filtration. The quality of the produced antibody samples was confirmed through analysis such as SEC-HPLC, CE-SDS, cIEF, ELISA, titer, and concentration.

[0045] Preparation Example 3. Analysis of the basic physical properties of HL161BKN Thermal stability, stability under accelerated conditions at different pH levels, and solubility tests were conducted on HL161BKN. For thermal stability, the Tm value was analyzed by DSC (Differential Scanning Calorimetry), and for accelerated condition experiments at various pH levels, the degree of aggregate and fragment formation was monitored during storage at 37°C for one month.

[0046] For DSC analysis of Tm values, equipment from the Osong Hi-tech Medical Industry Promotion Foundation was used. Experiments were conducted in the range of 25°C to 100°C, and the results were confirmed as shown in Figure 1. Typical DSC histograms for IgG1 types are shown, and curves corresponding to the CH domain and Fv domain are confirmed. HL161BKN showed a high Tm of 79.9°C and was analyzed to be very thermodynamically stable.

[0047] Furthermore, HL161BKN was prepared at a concentration of 10 mg / mL using sodium citrate buffer at pH 5.0, 6.0, 7.0, and 8.0. The degree of aggregate or fragment formation was then monitored using SEC-HPLC during storage at 37°C for 4 weeks. The results showed that aggregate or fragment formation increased with increasing pH (Figure 2).

[0048] Based on these results, HL161BKN was prepared at a concentration of 100 mg / mL using buffers with low or high concentrations at low pH levels of 5.0 and 6.0, and then stability tests were performed during storage at 40°C under accelerated conditions for 4 weeks. As a result, HL161BKN tended to be stable under low pH conditions of 5.0 to 6.0 and maintained high monomer levels overall under all conditions.

[0049] Furthermore, to confirm the solubility of HL161BKN, it was concentrated in six stages from 10 to 300 mg / mL, and samples were taken at each stage and observed visually. Analysis was then performed at A280 nm (concentration), A340 nm (turbidity), and SEC-HPLC purity. As a result, it was confirmed that the turbidity of HL161BKN gradually increased with increasing concentration, but the monomer purity remained almost unchanged up to a concentration of 268 mg / mL.

[0050] II. Selection of an optimized formulation for HL161BKN Preparation Example 1: Screening of optimized formulations for HL161BKN The purpose of this experiment is to select the active pharmaceutical ingredient and drug formulation for the development of a high-concentration subcutaneous administration product of HL161BKN. In this experiment, three production batches of HL161BKN-B005, HL161BKN-B018, and HL161BKN-B021 were used, and the reagents and equipment used are as follows (Table 3). [Table 3]

[0051] The equipment used for this formulation test is as follows (Table 4). [Table 4]

[0052] Preparation Example 2. Test Method Formulation selection tests for HL161BKN were broadly divided into excipient screening tests and excipient concentration screening tests. Specifically, the experiments were conducted using the same method as shown in Figure 3.

[0053] Example 1. Excipient screening Example 1.1. Excipient Test We selected 11 excipients frequently used in currently available antibody products: sucrose, D-trehalose, D-mannitol, D-sorbitol, L-arginine HCl, L-histidine HCl, L-histidine, L-glycine, polysorbate 20, polysorbate 80, and sodium chloride (NaCl). Specifically, we conducted excipient tests under 12 buffer conditions using 5 mM sodium citrate (pH 6.0) as the basal buffer (Table 5).

[0054] HL161BKN was concentrated to less than 1 mL using AMICON® (cutoff MW 30,000), and then the buffer was replaced with the corresponding buffer conditions to obtain a final concentration of 210 mg / mL. Samples were prepared in volumes of 0.3 to 0.5 mL for each condition and stored in 1.5 mL microcentrifuge tubes at 40°C for 4 weeks. Samples were sampled at weeks 0, 2, and 4 to evaluate changes in concentration (A280), turbidity (A340), and purity (SEC-HPLC, CEX-HPLC). [Table 5]

[0055] Example 1.2. Excipient combination test In Example 1.1, we investigated whether there was a synergistic effect when the five excipients selected (L-histidine, L-arginine hydrochloride, L-glycine, D-sorbitol, and D-mannitol) were combined. Specifically, we used a two-level factorial design (2) with DOE (Design of Experiments) software (Stat-ease DESIGN-EXPERT®, version 7.0). n-1 Seventeen conditional tests were planned (Table 6). Specifically, a total of 12 conditional tests were conducted: one under a 5 mM sodium citrate (pH 6.0) base buffer condition and one under 11 excipient combination conditions. Furthermore, the test results from Example 1.1 were used for conditional tests with only five excipients.

[0056] The test method was carried out in the same manner as in Example 1.1, and the osmotic pressures of the buffer and sample were further measured for each condition. Then, ANOVA analysis and effect values ​​were calculated using DOE software. [Table 6]

[0057] Example 1.3. Testing of further excipients Further analysis of excipients was conducted for the HL161BKN formulation test.

[0058] Further testing of L-methionine, known to reduce covalent aggregates, and testing of replacing the sodium citrate (pH 6.0) basal buffer, which may cause pain during injection, with histidine (pH 6.0) were performed. Furthermore, since the use of low-quality PSB20 (polysorbate 20) containing high levels of peroxides reduces antibody stability due to oxidation, this was replaced with high-quality PSB20 for the formulation. Subsequently, the effect on stirring stress was confirmed at a concentration of 0.02%, which is commonly used in formulations. Next, excipients that can reduce the viscosity of high-concentration antibody products were screened.

[0059] Example 1.3.1. Test of L-methionine and histidine basal buffer. To confirm the effect of L-methionine and whether the basal buffer, 5 mM sodium citrate (pH 6.0), could be replaced with histidine (pH 6.0), tests were conducted under the six conditions shown in Table 7. In the case of the histidine basal buffer, L-histidine and L-histidine hydrochloride were mixed and prepared to pH 6.0 before use. The test method was carried out in the same manner as in Example 1.1. [Table 7]

[0060] Example 1.3.2. Polysorbate 20 (PSB20) Test To confirm the protective effect of PSB20 against stirring stress, tests were conducted under the four conditions shown in Table 8. 0.5 mL of each sample, prepared in the same manner as in Example 1.1, was placed in a 1.5 mL microcentrifuge tube, mounted on a MyLab intelli mixer, and rotated at 10 rpm for one week at room temperature. Concentration, turbidity, and purity (SEC-HPLC) analysis were then performed. [Table 8]

[0061] Example 1.3.3. Screening of excipients for viscosity reduction To develop a high-concentration HL161BKN product for subcutaneous administration, eight excipients commonly known to reduce viscosity were tested. A total of nine excipient screening tests were performed using 50 mM histidine (pH 6.0) as the basal buffer (Table 9).

[0062] Samples were prepared in 1 mL in each condition, as in Example 1.1, and their viscosity at 25°C was measured. Furthermore, changes in concentration (A280) and purity (SEC-HPLC) for each condition were evaluated during a 4-week accelerated stability test. [Table 9]

[0063] Example 1.4. Excipient concentration screening Example 1.4.1.1 Excipient concentration screening Through Examples 1.1, 1.2, and 1.3, a 50 mM histidine (pH 6.0) basal buffer and three excipients (L-methionine, L-arginine hydrochloride, D-mannitol, and PSB20) were selected, and a two-level factor (2 n We planned DOE tests for the design and performed excipient concentration screening under a total of nine conditions (Table 10).

[0064] The experiment was carried out in the same manner as in Example 1.1, and changes in concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, and osmotic pressure were analyzed. [Table 10]

[0065] Example 1.4.2. Second excipient concentration screening. To determine the optimal concentration of L-arginine hydrochloride among the ultimately selected histidine basal buffer and two excipients (L-arginine hydrochloride and PSB20), accelerated stability tests were performed at 40°C for 4 weeks under four conditions using two batches of HL161BKNB018 and B021 (Table 11). The tests were performed in the same manner as in Example 1.1, and concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, osmotic pressure, and insoluble microparticles (microflow imaging; MFI) were analyzed. [Table 11]

[0066] Example 2. Results of excipient screening test Example 2.1. Results of excipient test To evaluate the effect of 11 excipients selected for excipient screening on the stability of HL161BKN samples, analyses of concentration, turbidity, purity, aggregates, fragments, and charge variants were performed at weeks 0, 2, and 4 under accelerated conditions of 40°C. These analyses selected five excipients (L-arginine hydrochloride, L-histidine, D-mannitol, L-glycine, and D-sorbitol) that showed good effects in suppressing aggregate and fragment formation.

[0067] Example 2.1.1. Results of concentration (A280) and turbidity (A340) analysis The HL161BKN concentration increased over four weeks (Table 12), which was presumed to be due to the evaporation of the buffer under accelerated conditions at 40°C. [Table 12]

[0068] On the other hand, turbidity did not increase or changed by less than 0.020 under most conditions. However, under PSB20 conditions, it was observed to be visibly cloudy, and a significant increase in turbidity (A340) was confirmed (Table 13). [Table 13]

[0069] Example 2.1.2. Results of aggregate and fragment analysis Using SEC-HPLC, the amount of aggregates and fragments increased under each excipient condition was compared and evaluated (Table 14). Compared to the basal buffer condition, L-arginine hydrochloride, L-histidine, L-histidine hydrochloride, D-mannitol, L-glycine, and D-sorbitol effectively suppressed aggregate formation. Furthermore, 0.2% PSB20, 0.2% PSB80, and NaCl actually increased aggregate formation. In addition, L-histidine, L-histidine hydrochloride, L-arginine hydrochloride, and L-glycine were excipients that inhibited fragment formation, while NaCl increased fragment formation (Table 14 and Figure 4). [Table 14]

[0070] Example 2.1.3. Results of charge variant analysis The change in charge variant patterns of HL161BKN under each excipient condition was examined using CEX-HPLC. No clear changes in charge variants were observed. However, the major peak decreased significantly under PSB20 and NaCl conditions (Figure 5). This was expected to be due to increased aggregate formation.

[0071] Example 2.2. Results of Excipient Combination Tests The effect of five excipient combinations selected in the excipient test of Example 1.1 on the stability of HL161BKN samples was evaluated. Specifically, an accelerated test at 40°C for 4 weeks was performed, and L-arginine hydrochloride, L-histidine, and D-mannitol were selected as effective inhibitors of aggregate and fragment formation.

[0072] To determine whether the excipients selected through the excipient screening test exhibited synergistic effects in combination, conditional tests were planned using Design of Experiments (DOE) software. A total of 12 conditional tests were conducted, including basic buffer conditions and 11 excipient combination conditions. The test results from Example 1.1 were used for conditional tests with only five excipients (Table 15).

[0073] ANOVA analysis of the changes in aggregate formation revealed excipients that statistically significantly inhibited aggregate formation (p<0.01). However, no excipients statistically significantly reduced fragment formation.

[0074] On the other hand, in the case of HL161BKN, the aggregate formation pattern differed depending on the combination of excipients, but there was no synergistic effect from the combination of excipients. The types of excipients were selected by referring to the ANOVA analysis and the comparative ranking of effect values. [Table 15]

[0075] Example 2.2.1. Analysis results of concentration (A280) and turbidity (A340) The concentration of HL161BKN increased over four weeks, which was presumed to be due to the evaporation of the buffer under accelerated conditions at 40°C (Table 16). On the other hand, turbidity did not increase or increased only slightly to below 0.054 (Table 17). [Table 16] [Table 17]

[0076] Example 2.2.2. Results of aggregate and fragment analysis Using SEC-HPLC, the amount of increased aggregates and fragments for each excipient combination was compared and evaluated (Table 18 and Figure 7). Furthermore, SEC-HPLC data for a total of 17 conditions, including the excipient-only test from Example 1.1, were analyzed by ANOVA. The results confirmed that L-arginine hydrochloride suppressed aggregate formation at a statistically significant level (p<0.01) (Figure 6), and no excipient suppressed fragment formation to a statistically significant level.

[0077] Furthermore, a comparison of the effectiveness values ​​against aggregation and fragmentation revealed that the formation of aggregates and fragments of HL161BKN was suppressed when L-histidine, D-mannitol, and D-sorbitol were used as excipients. However, there was no synergistic effect among the excipients. D-mannitol and D-sorbitol are isomers, and D-mannitol, which is frequently used, was selected (Figure 8). [Table 18]

[0078] Example 2.2.3. Results of charge variant analysis The pattern of charge variant changes in HL161BKN under different excipient combination conditions was examined using CEX-HPLC. As a result, no clear changes in charge variants (basic and acidic variants) were observed (Figure 9).

[0079] Example 2.2.4. Results of viscosity and osmotic pressure analysis Viscosity was measured for each excipient combination, and it was confirmed that the viscosity decreased upon the addition of L-arginine hydrochloride. Osmotic pressure increased with increasing number and concentration of added excipients. Specifically, the addition of 50 mM L-arginine hydrochloride, 50 mM L-histidine, and 100 mM L-glycine increased the osmotic pressure by approximately 100 mOsmol / kg, respectively; the addition of 200 mM D-mannitol increased the osmotic pressure by approximately 200 mOsmol / kg; and the addition of 250 mM D-sorbitol increased the osmotic pressure by approximately 250 mOsmol / kg (Table 19).

[0080] Generally, the osmotic pressure of subcutaneous injections is similar to the body's osmotic pressure (approximately 290 mOsmol / kg) and is controlled within a range of approximately 250 to 500 mOsmol / kg (PCT / EP2009 / 066675). Therefore, it was determined that the osmotic pressure of the formulation buffer should be controlled within a range of approximately 220 to 450 mOsmol / kg, taking into account the HL161BKN concentration. Accordingly, it was decided to proceed with subsequent tests for excipient concentration screening, taking the osmotic pressure range into consideration. [Table 19]

[0081] Example 2.3. Results of further excipient testing Further excipient tests were conducted. As a result, 50 mM histidine (pH 6.0) was used as the basal buffer, and L-methionine, which has an effect of suppressing aggregate formation, and 0.02% PSB20, which suppresses aggregate formation under stirring stress conditions, were selected as additional excipients.

[0082] Example 2.3.1. Results of L-methionine and histidine basal buffer tests 1) Analysis results of concentration (A280) and turbidity (A340) HL161BKN concentrations increased over four weeks, which was presumed to be due to the evaporation of the buffer under accelerated conditions at 40°C (Table 20). Turbidity did not increase or increased only slightly to less than 0.1 (Table 21). [Table 20] [Table 21]

[0083] 2) Results of aggregate and fragment analysis Compared to the basal buffer conditions, L-methionine showed a superior inhibitory effect on aggregate formation. Furthermore, there was no increase in aggregate formation with 0.02% PSB20. The results under the condition with L-histidine added as an excipient were similar to those under the condition using it as the basal buffer, and aggregate formation was reduced under the 50 mM histidine condition compared to the 10 mM histidine condition (Table 22 and Figure 10). [Table 22]

[0084] 3) Results of charge variant analysis CEX-HPLC analysis revealed no clear changes in charge variants (basic and acidic variants) (Figure 11).

[0085] Example 2.3.2. Results of the Polysorbate 20 Test 1) Results of concentration (A280) and turbidity (A340) analysis The stirring test at room temperature showed almost no change in concentration. The sample stirred without PSB20 appeared visibly white, and its turbidity could not be measured (Figure 12). On the other hand, the sample with PSB20 appeared slightly cloudy under stirring conditions, and its turbidity increased by approximately 0.227 (Table 23).

[0086] This confirmed that PSB20 has the effect of protecting high-concentration HL161BKN from stress caused by stirring. [Table 23]

[0087] 2) Results of aggregate and fragment analysis Aggregate formation increased with stirring stress, and it was confirmed that PSB20 has the effect of suppressing aggregate formation. On the other hand, there was no increase in fragments due to stirring stress (Table 24 and Figure 13). [Table 24]

[0088] Example 2.3.3. Results of screening of excipients for viscosity reduction 1) Results of concentration (A280) and viscosity analysis The HL161BKN concentration increased over four weeks (Table 25), which was presumed to be due to the evaporation of the buffer under accelerated conditions at 40°C. The addition of L-histidine hydrochloride, L-arginine hydrochloride, and L-glycine reduced viscosity compared to condition 1, but the effect was minor. On the other hand, the addition of L-lysine hydrochloride, NaCl, Na2SO4, and NH4Cl significantly increased viscosity (Table 26). [Table 25] [Table 26]

[0089] 2) Results of aggregate and fragment analysis Aggregates and fragments were analyzed by SEC-HPLC. The results showed that when L-histidine hydrochloride and L-arginine hydrochloride were added, aggregate formation was relatively low compared to condition 1, and no excipients were found to inhibit fragment formation (Table 27 and Figure 14). [Table 27]

[0090] Example 2.4. Results of excipient concentration screening Example 2.4.1. Results of the first excipient concentration screening To screen the concentrations of the histidine basal buffer (pH 6.0) and the three excipients (L-methionine, L-arginine hydrochloride, and D-mannitol) selected in Example 1.3 above in 0.02% PSB20, tests were performed under a total of nine conditions (Table 28). As a result, L-arginine hydrochloride and PSB20 were selected as excipients in addition to the histidine basal buffer. [Table 28]

[0091] 1) Results of concentration (A280) and turbidity (A340) analysis The HL161BKN concentration increased by approximately 15% over four weeks, which was estimated to be due to the evaporation of the buffer under accelerated conditions at 40°C (Table 29). Turbidity under each condition showed only a slight increase of approximately 0.048 on average (Table 30). [Table 29] [Table 30]

[0092] 2) Results of aggregate and fragment analysis SEC-HPLC analysis revealed no significant difference in the increase of aggregates and fragments under each excipient concentration condition. However, stability was high under the 50 mM histidine basal buffer condition (Condition 1), and aggregate formation decreased when 100 mM L-arginine hydrochloride was added compared to 50 mM. Furthermore, the amount of aggregates and fragments increased depending on the L-methionine concentration, and the presence or absence of added D-mannitol showed little difference from the basal buffer condition or only a slight increase (Table 31 and Figure 15). [Table 31]

[0093] 3) Results of charge variant analysis CEX-HPLC analysis revealed no clear changes in charge variants under each excipient condition (Figure 16).

[0094] 4) Results of viscosity and osmotic pressure analysis The viscosity and osmotic pressure were measured under various excipient concentration conditions. The results showed that adding L-arginine hydrochloride at a concentration of 100 mM was more effective in reducing viscosity than adding it at 50 mM, and most of these samples met the osmotic pressure criteria of 112 to 588 mOsmol / kg (Table 32). [Table 32]

[0095] Example 2.4.2.2nd Excipient Concentration Screening To select the optimal concentration of L-arginine hydrochloride, two batch samples of HL161BKN (HL161BKN B018 and HL161BKN B021) from the histidine basal buffer and two excipients selected in the first excipient concentration screening in Example 1.4.1 were evaluated for stability through concentration, turbidity, purity (aggregates and fragments), viscosity, osmotic pressure test, and suitability for subcutaneous injection (SC) administration under four conditions. As a result, 100 mM L-histidine, 100 mM L-arginine hydrochloride, 0.02% PSB20, and pH 6.0 were determined to be the final formulation of HL161BKN (Table 33). [Table 33]

[0096] 1) Concentration (A280) and turbidity (A340) analysis The HL161BKN concentration remained almost unchanged over four weeks, which we attribute to the effect of preventing buffer evaporation by sealing the 1.5 mL microcentrifuge tube with Parafilm and then storing it in a constant temperature and humidity chamber (Table 34). Furthermore, turbidity showed a slight increase to 0.102 or less under all conditions (Table 35). [Table 34] [Table 35]

[0097] 2) Aggregate and fragment analysis SEC-HPLC analysis showed no significant difference in the increase of aggregates and fragments under each excipient condition. However, the least aggregate formation was observed under condition 3 (100 mM L-histidine, 100 mM L-arginine HCl, 0.02% PSB20, pH 6.0) and condition 4 (50 mM L-histidine, 100 mM L-arginine HCl, 0.02% PSB20, pH 6.0) (Table 36 and Figure 17). [Table 36]

[0098] 3) Charge Variant Analysis CEX-HPLC analysis revealed no clear changes in charge variants (basic and acidic variants) under each excipient condition (Figure 18).

[0099] 4) Viscosity and osmotic pressure analysis Viscosity and osmotic pressure were measured under each excipient concentration condition. The viscosity was controlled to 11 to 16 cP, i.e., below the limit of 20 cP, which is the viscosity limit for subcutaneously administered products, and the osmotic pressure was 350 to 465 mOsmol / kg, meeting the standard of 250 to 500 mOsmol / kg (Table 37). [Table 37]

[0100] 5) Analysis of insoluble microparticles Insoluble microparticles were analyzed for each excipient condition sample of HL161BKN using microflow imaging (MFI) at the New Drug Development Support Center of the Osong Hi-tech Medical Industry Promotion Foundation.

[0101] The samples under each condition were diluted to 10 mg / mL, and the increase in the number of microparticles in the range of 5 μm to 100 μm was compared. As a result, the increase in the number of microparticles was smallest under condition 3 (Table 38 and Figure 19). [Table 38]

[0102] Example 3. Stability Test Catalent (USA) conducted 36-month long-term storage and accelerated stability tests for HL161BKN under the ultimately selected formulation conditions. To evaluate stability, the drug (DP) was stored in borosilicate glass vials with Teflon-coated rubber stoppers. The results confirmed the stability of the DP for 36 months under the selected formulation conditions, thereby ensuring its potential for development as an injectable formulation (Tables 39 and 40). [Table 39] [Table 40]

[0103] Example 4. Selection of final development candidate antibody formulation Since HL161BKN tends to be very stable at high concentrations in formulations, the aim was to confirm its potential for development in the form of an injectable drug for subcutaneous administration. When developing an injectable drug, it is known that a viscosity of 20 cP or less is suitable for subcutaneous administration in order to reduce pain and side effects at the injection site. Accordingly, HL161BKN was concentrated under nine different concentration conditions, and its viscosity was measured at 5°C and 25°C. The viscosity of an HL161BKN sample with a high concentration of 170 mg / mL was examined, and it was found to have a viscosity of 10 cP at 25°C, thus confirming that it is suitable for subcutaneous administration (Figure 20).

[0104] In the above formulation studies, it was confirmed that HL161BKN exhibits extremely stable properties even at high concentrations of 200 mg / mL or higher. As a result, it is expected that the development of a self-administered SC injection product will be possible in the future. Considering that all competing products are intravenous infusion type products, this will allow for differentiation by improving patient convenience.

[0105] Furthermore, since we confirmed that high concentrations of HL161BKN are stable in the formulation, we predicted that low concentrations of HL161BKN would also be stable in the formulation. Therefore, the formulation can be applied to HL161BKN at various concentrations.

Claims

1. (a) Anti-FcRn antibody or fragment thereof in a concentration of 50 mg / mL to 250 mg / mL (b) 50-250 mM arginine or its salt, (c) A buffer system selected from 50 to 250 mM histidine, and (d) 0.01-0.05% surfactant A pharmaceutical preparation having a pH of 5.0 to 6.0, including Pharmaceutical preparations that do not contain sucrose, Anti-FcRn antibody or fragment thereof A heavy chain variable region comprising H-CDR1 having the amino acid of SEQ ID NO: 5, H-CDR2 having the amino acid of SEQ ID NO: 6, and H-CDR3 having the amino acid of SEQ ID NO: 7; and Light chain variable region including L-CDR1 having the amino acid of SEQ ID NO: 8, L-CDR2 having the amino acid of SEQ ID NO: 9, and L-CDR3 having the amino acid of SEQ ID NO: 10 Pharmaceutical preparations, including those mentioned above.

2. The pharmaceutical formulation according to claim 1, wherein the surfactant is a polysorbate.

3. The pharmaceutical preparation according to claim 1, further comprising methionine.

4. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation is in the form of an injectable preparation.

5. The pharmaceutical formulation according to claim 1, wherein the viscosity of the pharmaceutical formulation is 20 cP or less.

6. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation has an osmotic pressure of 250 mOs / kg to 500 mOs / kg.

7. The pharmaceutical formulation according to claim 1, wherein the concentration of the anti-FcRn antibody or a fragment thereof is 80 mg / mL to 250 mg / mL.

8. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation is administered subcutaneously.

9. Pharmaceutical preparations, (a) Anti-FcRn antibody ranging from 50 mg / mL to 250 mg / mL, (b) 50 to 250 mM L-arginine or its hydrochloride, (c) 50 to 250 mM L-histidine buffer, and (d) containing 0.01 to 0.05% polysorbate 20, The pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation has a pH of 5.0 to 6.

0.

10. The pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation contains an anti-FcRn antibody, has increased stability, and the amount of aggregates and fragments of the anti-FcRn antibody in the formulation when stored for 6 months under accelerated conditions (25°C, 60% relative humidity) is 10% or less.

11. The pharmaceutical preparation according to claim 1, wherein the pharmaceutical preparation contains an anti-FcRn antibody and has increased stability, wherein the amount of aggregates and fragments of the anti-FcRn antibody in the preparation is 10% or less when stored for 36 months under long-term storage conditions (5°C).

12. The pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation contains an anti-FcRn antibody, has increased stability, and the amount of aggregates and fragments of the anti-FcRn antibody in the formulation when stored for 36 months under long-term storage conditions (5°C) is 5.0% or less.

13. A pharmaceutical preparation according to any one of claims 1 to 12, wherein the pharmaceutical preparation is for the treatment of an autoimmune disease selected from the group consisting of myasthenia gravis, thyroid eye disease, thermogenic autoimmune hemolytic anemia, neuromyelitis optica, immune thrombocytopenic purpura, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy, lupus nephritis, and membranous nephropathy.

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