Formulation for Anti-fcrn antibody

Abstract

One aspect of the present invention provides an aqueous pharmaceutical formulation with a pH of 4.0 to 8.0, comprising (a) an HL161 BKN antibody or a fragment thereof, (b) one or more additives selected from mannitol, sorbitol, arginine, histidine, glycine and salts thereof, (c) a buffer system selected from citrate or histidine, and (d) polysorbate. The HL161 BKN antibody present in the formulation has improved stability and is non-toxic, and thus has high industrial applicability.

Description

Formulations for anti-FCRn antibodies

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

[0002] The causes of autoimmune diseases have been studied for a long time from genetic, environmental, and immunological perspectives, but the precise cause remains unknown. Recent studies have revealed that many autoimmune diseases are caused by IgG-type autoantibodies. Indeed, research on the diagnosis and treatment of autoimmune diseases has widely established a correlation between the presence and reduction of disease-specific autoantibodies and therapeutic efficacy.

[0003] The first-line treatment for these autoimmune diseases is high-dose systemic steroid injections. However, in cases where symptoms are severe or difficult to control with steroids, high-dose intravenous immunoglobulin (IVIG) or plasmapheresis are used. High-dose steroids are often ineffective or can cause serious side effects with repeated use. Furthermore, IVIG and plasmapheresis are expensive and carry various side effects and risks of infection, making the development of therapeutics in this area urgently needed.

[0004] Meanwhile, a treatment for autoimmune diseases using FcRn antibodies is currently being studied (Korean Patent Publication No. 10-2014-0147606). This antibody is a novel drug mechanism that blocks FcRn (Neonatal Fc Receptor), which is involved in IgG recycling, thereby increasing the rate of IgG clearance (catabolism) in the body and thereby reducing autoantibodies, thereby treating the disease. These anti-FcRn antibodies are expected to solve the problems of existing treatments.

[0005] However, in order to apply these antibodies to severe autoimmune diseases such as pemphigus bulgaris, neuromyelitis optica, and myasthenia gravis, which are caused by the production of autoantibodies against autoantigens in the body, an optimized formulation is required according to the formulation and administration method optimized for these antibodies.

[0006] The present inventors conducted research to develop a formulation optimized for an anti-FcRn antibody for treating severe autoimmune diseases, and as a result, developed a buffer and formulation optimized for HL161BKN, an anti-FcRn antibody.

[0007] To achieve the above object, one aspect of the present invention provides a pharmaceutical preparation 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.

[0008] A pharmaceutical formulation having a pH of 4.0 to 8.0 comprising (a) an anti-FcRn antibody or a fragment thereof according to the present invention, (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 is a pharmaceutical composition optimized for HL161BKN, and it was confirmed that the stability of HL161BKN is improved in the formulation.

[0009] Figure 1 shows the analysis of the thermal stability of HL161BKN using DSC (Differential Scanning Calorimetry).

[0010] Figure 2 shows the 4-week accelerated stability of HL161BKN under sodium citrate-phosphate buffer pH 5.0, 6.0, 7.0, and 8.0 conditions.

[0011] Figure 3 is a schematic diagram of the process for studying the formulation of HL161BKN.

[0012] Figure 4 shows the results of the excipient test, confirming the change in the amount of aggregates and fragments of HL161BKN (210 mg / mL) produced depending on the excipient.

[0013] Figure 5 shows the charge variants of HL161BKN (210 mg / mL) according to each excipient condition, analyzed using CEX-HPLC.

[0014] Figure 6 shows the results of analysis of variance (ANOVA) on the change in aggregate amount of HL161BKN according to the combination of excipients.

[0015] Figure 7 shows the changes in the amount of aggregates and fragments of HL161BKN (210 mg / mL) produced according to the combination of excipients.

[0016] Figure 8 shows the results of a design of experiment (DOE) analysis of the changes in the aggregates and fragments of HL161BKN according to the combination of excipients.

[0017] Figure 9 shows the charge variants of HL161BKN according to the combination of excipients, analyzed using CEX-HPLC.

[0018] Figure 10 shows the changes in the amount of aggregates and fragments of HL161BKN (210 mg / mL) in additional excipient tests.

[0019] Figure 11 shows the charge variants of HL161BKN according to excipient conditions in an additional excipient test, analyzed using CEX-HPLC.

[0020] Figure 12 shows the effect of the presence or absence of PSB20 on the stirring stress of HL161BKN (210 mg / mL).

[0021] Figure 13 shows the change in monomer of HL161BKN depending on the presence or absence of PSB20 in a stirring test.

[0022] Figure 14 shows the change in the amount of aggregates and fragments of HL161BKN (210 mg / mL) in the viscosity reducing excipient test.

[0023] Figure 15 shows the changes in aggregates and fragments of HL161BKN (210 mg / mL) in the first excipient screening.

[0024] Figure 16 shows the charge variants of HL161BKN according to the primary excipient screening conditions, analyzed using CEX-HPLC.

[0025] Figure 17 shows the changes in aggregates and fragments of HL161BKN (210 mg / mL) in the secondary excipient screening.

[0026] Figure 18 shows the charge variants of HL161BKN according to secondary excipient conditions, analyzed using CEX-HPLC.

[0027] Figure 19 shows the change in the number of sub-visible particles of HL161BKN.

[0028] Figure 20 shows the results of confirming the viscosity according to the concentration of HL161BKN in the HL161BKN formulation. At this time, when HL161BKN was 170 mg / mL, it was confirmed to be 10 cP.

[0029] One aspect of the present invention provides 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 salts thereof, (c) a buffer system selected from citrate or histidine, and (d) a surfactant.

[0030] Additionally, the pharmaceutical preparation may further comprise methionine. Additionally, the pharmaceutical preparation may further comprise a sugar such as sucrose or trehalose.

[0031] The above buffer system refers to a buffer that is resistant to changes in pH due to its conjugate acid-base components.

[0032] The term "histidine buffer" as used herein refers to a buffer containing histidine ions. Here, specific examples of the histidine buffer may be any one selected from the group consisting of a histidine chloride buffer, a histidine acetate buffer, a histidine phosphate buffer, and a histidine sulfate buffer, but are not limited thereto.

[0033] The term "citrate buffer" as used herein refers to a buffer containing citrate ions. Here, specific examples of the citrate buffer may be any one selected from the group consisting of, but not limited to, a citrate-sodium buffer, a citrate-potassium buffer, a citrate-calcium buffer, and a citrate-magnesium buffer.

[0034] The term "surfactant" as used herein refers to a pharmaceutically acceptable excipient used to protect protein formulations against mechanical stresses such as agitation and shearing. Specific examples 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, lauryl amidopropyl-betaine, cocaramidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitoylamidopropyl-betaine, It may be any one surfactant selected from the group consisting of isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitoylamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocayl, sodium methyl oleyl-taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol. In this case, the surfactant may preferably be polysorbate 20.

[0035] Additionally, the pharmaceutical preparation may be an aqueous preparation, and preferably an injectable liquid preparation.

[0036] At this time, the anti-FcRn antibody may be HL161BKN.

[0037] The above HL161BKN comprises a heavy chain variable region comprising an H-CDR1 having an amino acid sequence of SEQ ID NO: 5, an H-CDR2 having an amino acid sequence of SEQ ID NO: 6, and an H-CDR3 having an amino acid sequence of SEQ ID NO: 7, and a light chain variable region comprising an L-CDR1 having an amino acid sequence of SEQ ID NO: 8, an L-CDR2 having an amino acid sequence of SEQ ID NO: 9, and an L-CDR3 having an amino acid sequence of SEQ ID NO: 10.

[0038] Additionally, the HL161BKN may comprise the heavy and light chain regions described in Table 1 below. Additionally, the heavy and light chains may be encoded by the nucleic acids described in Table 2.

[0039]

[0040] The glycosylation sites of the above antibody are as follows: Asn301, N-glycan (G0F, G1F, G0-GlcNac, Man5).

[0041]

[0042] At this time, the pharmaceutical preparation may be characterized by having a viscosity of 20 cP or less. Specifically, the pharmaceutical preparation may have a viscosity of 1 cP to 20 cP. In addition, the pharmaceutical preparation 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.

[0043] Additionally, the pharmaceutical formulation may be characterized by having an osmolality of 250 mOs / kg to 500 mOs / kg. Specifically, the pharmaceutical formulation may have an osmolality of 300 mOs / kg to 450 mOs / kg or 350 mOs / kg to 400 mOs / kg. In addition, the pharmaceutical preparation may have an O2 concentration of about 250 mOs / kg, about 260 mOs / kg, about 270 mOs / kg, about 280 mOs / kg, about 290 mOs / kg, about 300 mOs / kg, about 310 mOs / kg, about 320 mOs / kg, about 330 mOs / kg, about 340 mOs / kg, about 350 mOs / kg, about 360 mOs / kg, about 370 mOs / kg, about 380 mOs / kg, about 390 mOs / kg, about 400 mOs / kg, about 410 mOs / kg, about 420 mOs / kg, about 430 mOs / kg, about 440 mOs / kg, about 450 mOs / kg, about 460 mOs / kg, about 470 mOs / kg, about 480 mOs / kg, about 490 mOs / kg, or about 500 mOs / kg.

[0044] Additionally, the pharmaceutical formulation may comprise HL161BKN at a concentration of 50 mg / mL to 250 mg / mL. Specifically, the pharmaceutical formulation may comprise 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. Additionally, the pharmaceutical formulation may comprise HL161BKN at 120 mg / mL to 230 mg / mL, 150 mg / mL to 220 mg / mL, or 180 mg / mL to 210 mg / mL. Additionally, the pharmaceutical formulation may comprise HL161BKN at about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, or about 250 mg / mL.

[0045] Additionally, 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. Additionally, the pharmaceutical formulation may have a pH of about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about pH 6.0, about pH 6.1, about pH 6.2, about pH 6.3, about pH 6.4, about pH 6.5, about pH 6.6, about pH 6.7, about pH 6.8, about pH 6.9, or about pH 7.0.

[0046] Additionally, the additive may be included in an amount of 10 mM to 400 mM. At this time, the additive may be used alone as mannitol, sorbitol, arginine, histidine, or glycine, or two or more may be used in combination. Specifically, the additive may be included in an amount of 10 mM to 400 mM, 20 mM to 300 mM, 50 mM to 250 mM, or 100 mM to 150 mM, respectively. Specifically, the additives may be included in an amount of about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, respectively.

[0047] In addition, two of the additives may be used in combination. In one specific example, the additive may include mannitol and sorbitol. In one specific example, the additive may include mannitol and arginine. In one specific example, the additive may include mannitol and histidine. In one specific example, the additive may include mannitol and glycine. In one specific example, the additive may include sorbitol and arginine. In one specific example, the additive may include sorbitol and histidine. In one specific example, the additive may include sorbitol and glycine. In one specific example, the additive may include arginine and histidine. In one specific example, the additive may include arginine and glycine. In one specific example, the additive may include histidine and glycine. In this case, each additive may be included in the pharmaceutical preparation at the concentration described above.

[0048] One specific example 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 an 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% of polysorbate 20.

[0049] At this time, the anti-FcRn antibody is as described above. In addition, the pharmaceutical preparation may be an aqueous preparation or an injectable liquid preparation. In addition, the pharmaceutical preparation described above may be characterized as being administered subcutaneously.

[0050] Furthermore, the pharmaceutical formulation was confirmed to be highly stable under accelerated conditions. Specifically, the content of aggregates and fragments may be approximately 10% or less in a 6-month test under accelerated conditions (25°C, 60% relative humidity). Furthermore, 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 5.5% or less, or approximately 5.0% or less.

[0051] In addition, it was confirmed that the pharmaceutical preparation is very stable even under long-term storage conditions. Specifically, the content of aggregates and fragments may be about 10% or less under conditions of 5°C and 36 months. In addition, the content of aggregates and fragments under the above conditions may be about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1.8% or less, about 1.5% or less, or about 1.2% or less.

[0052] In addition, the pharmaceutical preparation may be used for the treatment of an autoimmune disease. At this time, the autoimmune disease may be any one selected from the group consisting of Myasthenia Gravis (MG), Thyroid Eye Disease (TED), Warm 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).

[0053] Screening of formulations optimized for HL161BKN

[0054] HL161BKN was manufactured at a concentration of approximately 210 mg / mL for each condition and stored for 4 weeks under accelerated conditions at 40℃. The stability of the sample and suitability for subcutaneous administration were evaluated by performing analyses such as concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, osmotic pressure, and insoluble fine particles (MFI).

[0055] First, a screening test was conducted on 11 excipients frequently used in existing antibody products. The results confirmed that L-histidine, L-arginine hydrochloride, L-glycine, D-sorbitol, and D-mannitol were effective in reducing aggregate formation.

[0056] The synergistic effect of combining five selected excipients was tested. As a result, L-arginine hydrochloride was confirmed to statistically significantly reduce aggregate formation (p<0.01).

[0057] Based on the comparison of effect values ​​for aggregates and fragmentation, L-histidine and D-mannitol were selected as excipients. Furthermore, L-methionine, which demonstrated a reduced aggregation formation effect through additional excipient screening, was additionally selected. Furthermore, the feasibility of histidine as a basic buffer was confirmed. Furthermore, high-purity 0.02% polysorbate 20 was selected for its ability to suppress aggregate formation caused by stirring stress that may occur during product storage and transport.

[0058] Next, a primary concentration screening test was performed with the selected histidine base buffer and L-arginine hydrochloride, D-mannitol, and L-methionine excipients. The concentration of PBS20 was fixed at 0.02% and the test was performed. As a result, high stability was confirmed under the condition of 50 mM histidine base buffer without excipients. In addition, there was no significant difference in the increase in aggregates and fragments according to the excipient condition, but aggregate formation decreased as the concentration of added L-arginine hydrochloride increased.

[0059] On the other hand, it was confirmed that there was little difference according to the presence or absence of D-mannitol and the concentration of L-methionine. Therefore, L-arginine hydrochloride, 0.02% PSB20 in the L-histidine basic buffer was selected as the HL161BKN formulation.

[0060] Finally, a secondary concentration screening was performed using two batches of HL161BKN to determine the optimal concentrations of L-histidine and L-arginine hydrochloride. As a result, no significant differences were observed in the increase in aggregates and fragments according to excipient concentration conditions, and viscosity and osmolality measurements under all conditions were confirmed to be below 20 cP and 250–500 mOsmol / kg, respectively, suitable for subcutaneous administration.

[0061] In addition, as a result of performing insoluble particulate analysis using micro flow imaging (MFI) for each condition sample, the condition with the smallest increase in the number of insoluble particulates (sub-visible particles) was confirmed, and the final formulation was selected by evaluating the purity (aggregates and fragments), viscosity, stability through osmotic pressure tests, and suitability for subcutaneous injection (SC) administration.

[0062] In conclusion, the formulation for nonclinical and phase 1 clinical trials of HL161BKN was selected as approximately 100 mM L-histidine, approximately 100 mM L-arginine hydrochloride, and approximately 0.02% polysorbate 20 (pH 6.0).

[0063] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0064] I. Manufacturing of HL161BKN

[0065] Manufacturing Example 1. Preparation of gene construct and vector for manufacturing HL161 antibody.

[0066] To manufacture HL161BKN, a polynucleotide having a nucleic acid sequence of SEQ ID NO: 3 encoding a heavy chain comprising the amino acid of SEQ ID NO: 1 was loaded into the pCHO 1.0 vector (Life Technologies). In addition, a polynucleotide having a nucleic acid sequence of SEQ ID NO: 4 encoding a light chain comprising the amino acid of SEQ ID NO: 2 was loaded into the pCHO 1.0 vector.

[0067] Manufacturing Example 2. Preparation and production of HL161BKN cell line

[0068] The expression vector constructed in Manufacturing Example 1 was used to transform CHO-S cells, and the final production cell line was constructed after selection with methotrexate and puromycin. The constructed cell line was stored in a cell bank and used for production of HL161BKN. Antibody production was performed by culturing the cells for approximately 15 days in a bioreactor containing culture medium (Dynamis medium + 8 mM L-glutamine) by adding supplementary medium (EFB+) every two days, and then collecting the supernatant. After that, Protein A column was performed, and viral inactivation was performed at low pH, followed by anion exchange chromatography (AEX) and cation exchange chromatography (CEX). Subsequently, the sample was purified by concentration and buffer exchange (Ultrafiltration / Diafiltration) and then sterile filtration. The quality of the produced antibody sample was confirmed through analyses such as SEC-HPLC, CE-SDS, cIEF, ELISA, Potency, and Concentration.

[0069] Manufacturing Example 3. Basic Characteristics Analysis of HL161BKN

[0070] We conducted thermal stability and solubility tests on HL161BKN under accelerated pH conditions. Thermal stability was analyzed by Tm value using differential scanning calorimetry (DSC). For the pH-dependent accelerated condition experiment, the degree of aggregate and fragment formation was monitored while the sample was stored at 37°C for one month.

[0071] DSC for Tm value analysis was performed using equipment from the Osong Advanced Medical Industry Promotion Foundation. Experiments were conducted over a temperature range of 25°C to 100°C, and the results are shown in Figure 1. A typical DSC histogram of the IgG1 type was shown, and curves according to the CH domain and Fv domain were identified. HL161BKN exhibited a high Tm of 79.9°C, indicating that it is thermodynamically very stable.

[0072] Additionally, HL161BKN was prepared at a concentration of 10 mg / mL using sodium citrate-phosphate buffers at pH 5.0, 6.0, 7.0, and 8.0. The resulting solution was stored at 37°C for 4 weeks and the degree of aggregate or fragment formation was determined using SEC-HPLC. As a result, the higher the pH, the more aggregate or fragment formation occurred (Fig. 2).

[0073] Based on these results, HL161BKN was manufactured at 100 mg / mL using low- and high-concentration buffers with low pH values ​​of 5.0 and 6.0, and then stored for 4 weeks under accelerated conditions at 40°C to conduct a stability test. As a result, HL161BKN showed a tendency to be stable under low pH conditions of pH 5.0 to 6.0, and maintained a high level of monomer overall under all conditions.

[0074] In addition, to confirm the solubility of HL161BKN, samples were collected at each stage while concentrating in 6 steps from 10 to 300 mg / mL, and visual observation, A280 nm (concentration), A340 nm (turbidity), and SEC-HPLC purity analysis were performed. As a result, it was confirmed that the turbidity of HL161BKN gradually increased as the concentration increased, but there was almost no change in monomer purity up to a concentration of 268 mg / mL.

[0075] II. Selection of formulation optimized for HL161BKN

[0076] Preparation Example 1. Screening of formulation optimized for HL161BKN

[0077] The purpose of this experiment was to select the formulation of the drug substance and product for the development of a high-concentration subcutaneous administration product of HL161BKN. This experiment used three production batches, HL161BKN-B005, HL161BKN-B018, and HL161BKN-B021, and the reagents and equipment used are as follows (Table 3).

[0078]

[0079] The devices used for this formulation test are as follows (Table 4).

[0080]

[0081] Preparation Example 2. Test Method

[0082] The formulation selection test for HL161BKN was broadly divided into excipient screening and excipient concentration screening tests. Specifically, the experiment was conducted using the method shown in Figure 3.

[0083] Example 1. Excipient screening

[0084] Example 1.1. Excipient Test

[0085] Eleven types of excipients frequently used in currently commercially available antibody products were selected: 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, excipients tests were performed under 12 buffer conditions using 5 mM sodium citrate (pH 6.0) as the base buffer (Table 5).

[0086] HL161BKN by Amicon ® (Cut off MW. 30,000) was used to concentrate to less than 1 mL, and then the buffer was exchanged under the corresponding buffer conditions to prepare a final concentration of 210 mg / mL. 0.3 to 0.5 mL of each sample was prepared for each condition, placed in a 1.5 mL microcentrifuge tube, and stored 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).

[0087]

[0088] Example 1.2. Excipient combination test

[0089] The synergistic effect was confirmed when combining the five excipients (L-histidine, L-arginine hydrochloride, L-glycine, D-sorbitol, and D-mannitol) selected in Example 1.1. Specifically, DOE (design of experiments) software (Stat-ease Design-Expert ® , version 7.0) using 2 level factorial(2n-1 ) design, 17 condition tests were planned (Table 6). Specifically, a total of 12 condition tests were performed, including the basic buffer condition of 5 mM sodium citrate (pH 6.0) and 11 excipient combination conditions. In addition, the test results of Example 1.1 were used for the 5 excipient-only condition tests.

[0090] The test method was performed in the same manner as in Example 1.1, and additionally, the osmotic pressure for the buffer and sample for each condition was measured, and the DOE Software was used to perform ANOVA analysis and calculate the effects value.

[0091]

[0092] Example 1.3. Additional excipient testing

[0093] Additional excipient analysis was conducted for the HL161BKN formulation test.

[0094] Additional testing was performed with L-methionine, known to reduce covalent aggregates, and a test was performed to change the basic buffer from sodium citrate (pH 6.0), which can cause pain upon injection, to histidine (pH 6.0). Furthermore, because low-quality PSB20 (Polysorbate 20) with a high peroxide content reduces antibody stability due to oxidation, a high-quality PSB20 for formulation was used. The formulation was then tested for its effect on agitation stress at a commonly used concentration of 0.02%. Furthermore, excipients capable of reducing the viscosity of high-concentration antibody products were screened.

[0095] Example 1.3.1. L-Methionine and Histidine Basal Buffer Test

[0096] To determine the effect of L-methionine and whether the basic buffer, 5 mM sodium citrate (pH 6.0), can be changed to histidine (pH 6.0), a test was conducted under the six conditions in Table 7. For the histidine basic buffer, L-histidine and L-histidine hydrochloride were mixed to prepare a pH of 6.0 and used. The test method was the same as in Example 1.1.

[0097]

[0098] Example 1.3.2. Polysorbate 20 (PSB20) test

[0099] To confirm the protective effect of PSB20 against agitation stress, a test was performed under the four conditions in Table 8. 0.5 mL of the samples prepared in the same manner as in Example 1.1 were placed in 1.5 mL microcentrifuge tubes, mounted on a MyLab intelli mixer, and rotated at 10 rpm for one week at room temperature, followed by concentration, turbidity, and purity (SEC-HPLC) analysis.

[0100]

[0101] Example 1.3.3. Screening of excipients for viscosity reduction

[0102] To develop a high-concentration product for subcutaneous administration of HL161BKN, eight excipients known to generally reduce viscosity were tested. A total of nine excipient screening tests were conducted using 50 mM histidine (pH 6.0) as the base buffer (Table 9).

[0103] Samples were prepared in 1 mL amounts for each condition using the same method as Example 1.1, and their viscosity was measured at 25°C. In addition, changes in concentration (A280) and purity (SEC-HPLC) for each condition were evaluated during a 4-week accelerated stability test.

[0104]

[0105] Example 1.4. Excipient Concentration Screening

[0106] Example 1.4.1. Primary excipient concentration screening

[0107] Among the 50 mM histidine (pH 6.0) basic buffer and 4 excipients (L-methionine, L-arginine hydrochloride, D-mannitol, PSB20) selected through Examples 1.1, 1.2 and 1.3, 3 types (L-methionine, L-arginine hydrochloride, D-mannitol) were tested at 2 level factorial (2 n ) design, a DOE test was planned and excipient concentration screening was performed under a total of nine conditions (Table 10).

[0108] The experiment was performed in the same manner as in Example 1.1, and the concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, and osmotic pressure changes were analyzed.

[0109]

[0110] Example 1.4.2. Secondary excipient concentration screening

[0111] Among the finally selected histidine base buffer and two excipients (L-arginine hydrochloride, PSB20), an accelerated stability test at 40°C for 4 weeks was performed under four conditions using two batches of HL161BKN B018 and B021 to determine the optimal concentration of L-arginine hydrochloride (Table 11). The test was performed in the same manner as in Example 1.1, and the concentration (A280), turbidity (A340), purity (SEC-HPLC, CEX-HPLC), viscosity, osmotic pressure, and insoluble particulate matter (Micro flow imaging; MFI) were analyzed.

[0112]

[0113] Example 2. Results of excipient screening test

[0114] Example 2.1. Results of excipient test

[0115] To evaluate the effects of 11 excipients selected for excipient screening on the stability of HL161BKN samples, concentration, turbidity, purity, aggregates, fragments, and charge variants were analyzed at weeks 0, 2, and 4 under accelerated conditions at 40°C. Through these analyses, five excipients (L-arginine hydrochloride, L-histidine, D-mannitol, L-glycine, and D-sorbitol) that were effective in inhibiting the formation of aggregates and fragments were selected.

[0116] Example 2.1.1. Results of concentration (A280) and turbidity (A340) analysis

[0117] The HL161BKN concentration increased over 4 weeks (Table 12), which was presumed to be due to buffer evaporation under accelerated conditions at 40°C.

[0118]

[0119] In contrast, under most conditions, turbidity did not increase or showed a change of less than 0.020. However, under the PSB20 condition, a visually cloudy change was observed, and turbidity (A340) also increased significantly (Table 13).

[0120]

[0121] Example 2.1.2. Results of aggregate and fragment analysis

[0122] The increase in aggregates and fragments under each excipient condition was compared and evaluated using SEC-HPLC (Table 14). Compared to the basic buffer condition, L-arginine hydrochloride, L-histidine, L-histidine hydrochloride, D-mannitol, L-glycine, and D-sorbitol effectively inhibited aggregate formation. In addition, 0.2% PSB20, 0.2% PSB80, and NaCl actually increased aggregate formation. In addition, the excipients that inhibited fragment formation were L-histidine, L-histidine hydrochloride, L-arginine hydrochloride, and L-glycine, while NaCl increased fragment formation (Table 14 and Fig. 4).

[0123]

[0124] Example 2.1.3. Results of charge variant analysis

[0125] Using CEX-HPLC, we examined the charge variant changes of HL161BKN under various excipient conditions. No significant changes in charge variants were observed. However, the main peak significantly decreased under PSB20 and NaCl conditions (Fig. 5). This was expected to be due to an increase in aggregates.

[0126] Example 2.2. Results of excipient combination test

[0127] The stability effects of HL161BKN samples were evaluated by combinations of five excipients selected from the excipient test in Example 1.1. Specifically, an accelerated test was conducted at 40°C for 4 weeks, and L-arginine hydrochloride, L-histidine, and D-mannitol were selected to effectively suppress the formation of aggregates and fragments.

[0128] To determine whether the excipients selected through excipient screening tests exhibited synergistic effects in combination, conditional tests were planned using design of experiments (DOE) software. A total of 12 conditional tests were conducted, including the basic buffer condition and 11 excipient combination conditions. The test results from Example 1.1 were used for the five excipients tested individually (Table 15).

[0129] When the results of the comparison of aggregate changes were analyzed by ANOVA, we identified excipients that reduced aggregate formation to a statistically significant level (p<0.01). However, no excipients were found to statistically significantly reduce fragment formation.

[0130] In contrast, for HL161BKN, aggregate formation patterns varied depending on the excipient combination, but there was no synergistic effect by excipient combination. The excipient types were selected with reference to the ANOVA analysis and comparative ranking of effect values.

[0131]

[0132] Example 2.2.1. Results of concentration (A280) and turbidity (A340) analysis

[0133] The HL161BKN concentration increased over a 4-week period, which was presumed to be due to buffer evaporation under accelerated conditions at 40°C (Table 16). On the other hand, turbidity showed no increase or a minimal increase of less than 0.054 (Table 17).

[0134]

[0135]

[0136] Example 2.2.2. Results of aggregate and fragment analysis

[0137] The increase in aggregates and fragments under each excipient combination condition was compared and evaluated using SEC-HPLC (Table 18 and Figure 7). In addition, the SEC-HPLC data for a total of 17 conditions, including the excipient-only condition test of Example 1.1, were analyzed by ANOVA. As a result, L-arginine hydrochloride was confirmed to reduce aggregate formation to a statistically significant level (p<0.01) (Figure 6), and no excipient reduced fragment formation to a statistically significant level.

[0138] In addition, as a result of comparing the effect values ​​for aggregation and fragmentation, it was confirmed that the production of aggregates and fragments of HL161BKN decreased when L-histidine, D-mannitol, and D-sorbitol were used as excipients. However, there was no synergistic effect between the excipients. D-mannitol and D-sorbitol are isomers, and among the two, D-mannitol was selected as it is more frequently used (Fig. 8).

[0139]

[0140] * A: 50 mM L-arginine hydrochloride, H: 50 mM L-histidine, G: 100 mM L-glycine, M: 200 mM D-mannitol, S: 250 mM D-sorbitol

[0141] Example 2.2.3. Results of charge variant analysis

[0142] The charge variant changes of HL161BKN according to the excipient combination conditions were examined using CEX-HPLC. As a result, no significant changes in charge variants (basic and acidic variants) were observed (Fig. 9).

[0143] Example 2.2.4. Results of viscosity and osmotic pressure analysis

[0144] As a result of measuring the viscosity under each excipient combination condition, it was confirmed that the viscosity tended to decrease when L-arginine hydrochloride was added. The osmotic pressure increased as the number and concentration of added excipients increased. Specifically, it was confirmed that the osmotic pressure increased by approximately 100 mOsmol / kg when 50 mM L-arginine hydrochloride, 50 mM L-histidine, and 100 mM L-glycine were added, by approximately 200 mOsmol / kg when 200 mM D-mannitol was added, and by approximately 250 mOsmol / kg when 250 mM D-sorbitol was added (Table 19).

[0145] Typically, the osmolality of subcutaneous injections is similar to the body's osmolality (approximately 290 mOsmol / kg) and is controlled within a range of approximately 250 to 500 mOsmol / kg (PCT / EP2009 / 066675). Therefore, considering the concentration of HL161BKN, it was determined that the osmolality of the formulation buffer should be controlled within a range of approximately 220 to 450 mOsmol / kg. Therefore, considering the osmolality range, it was decided to conduct a subsequent excipient concentration screening test.

[0146]

[0147] * A: 50 mM L-arginine hydrochloride, H: 50 mM L-histidine, G: 100 mM L-glycine, M: 200 mM D-mannitol, S: 250 mM D-sorbitol

[0148] Example 2.3. Results of additional excipient testing

[0149] Additional excipient tests were performed. As a result, 50 mM histidine (pH 6.0) was used as the basic buffer, and L-methionine, which has an inhibitory effect on aggregate formation, and 0.02% PSB20, which inhibits aggregate formation under stirring stress conditions, were selected as additional excipients.

[0150] Example 2.3.1. Results of L-methionine and histidine basic buffer tests

[0151] 1) Results of concentration (A280) and turbidity (A340) analysis

[0152] The HL161BKN concentration increased over a 4-week period, which was presumed to be due to buffer evaporation under accelerated conditions at 40°C (Table 20). Turbidity showed no increase or a minimal increase of less than 0.1 (Table 21).

[0153]

[0154]

[0155] 2) Results of aggregate and fragment analysis

[0156] Compared to the basic buffer condition, L-methionine showed a superior effect in suppressing aggregate formation. In addition, there was no increase in aggregate formation by 0.02% PSB20. In addition, the results of the conditions in which L-histidine was added as an excipient and the conditions in which it was used as a basic buffer were similar, and aggregate formation was reduced in the 50 mM histidine condition compared to the 10 mM histidine condition (Table 22 and Figure 10).

[0157]

[0158] 3) Results of charge variant analysis

[0159] As a result of CEX-HPLC analysis, no changes in distinct charge variants (basic and acidic variants) could be observed (Fig. 11).

[0160] Example 2.3.2. Results of the Polysorbate 20 Test

[0161] 1) Results of concentration (A280) and turbidity (A340) analysis

[0162] Stirring tests at room temperature revealed minimal concentration changes. Samples stirred without PSB20 appeared visually white, making turbidity unmeasurable (Fig. 12). In contrast, samples with PSB20 appeared slightly cloudy when stirred, and turbidity increased by approximately 0.227 (Table 23).

[0163] Through this, it was confirmed that PSB20 has a protective effect against the stress caused by stirring of high concentration HL161BKN.

[0164]

[0165] 2) Results of aggregate and fragment analysis

[0166] Aggregate formation was increased by stirring stress, and the inhibitory effect of PSB20 on aggregate formation was confirmed. On the other hand, no increase in fragments was observed by stirring stress (Table 24 and Fig. 13).

[0167]

[0168] Example 2.3.3. Results of screening excipients for viscosity reduction

[0169] 1) Results of concentration (A280) and viscosity analysis

[0170] The HL161BKN concentration increased over 4 weeks (Table 25), which was presumed to be due to buffer evaporation under accelerated conditions at 40℃. When L-histidine hydrochloride, L-arginine hydrochloride, and L-glycine were added, the viscosity decreased compared to Condition 1, but the effect was minimal. On the other hand, L-lysine hydrochloride, NaCl, Na2SO4, and NH4Cl showed a significant increase in viscosity (Table 26).

[0171]

[0172]

[0173] 2) Results of aggregate and fragment analysis

[0174] Aggregates and fragments were analyzed using SEC-HPLC analysis. As a result, compared to Condition 1, when L-histidine hydrochloride and L-arginine hydrochloride were added, relatively fewer aggregates were generated, and no excipients were found to reduce fragment production (Table 27 and Figure 14).

[0175]

[0176] Example 2.4. Results of Excipient Concentration Screening

[0177] Example 2.4.1. Results of primary excipient concentration screening

[0178] In order to screen the concentrations of three excipients (L-methionine, L-arginine hydrochloride, D-mannitol) in the histidine basic buffer (pH 6.0) and 0.02% PSB20 selected in Example 1.3 above, tests were performed under a total of nine conditions (Table 28). As a result, L-arginine hydrochloride and PSB20 were selected as excipients in the histidine basic buffer.

[0179]

[0180] 1) Results of concentration (A280) and turbidity (A340) analysis

[0181] The HL161BKN concentration increased by approximately 15% over 4 weeks, which was presumed to be due to buffer evaporation 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).

[0182]

[0183]

[0184] 2) Results of aggregate and fragment analysis

[0185] SEC-HPLC analysis results showed no significant difference in the amount of aggregates and fragments increased according to each excipient concentration condition. However, stability was higher under the basic buffer condition of 50 mM histidine (condition 1), and aggregate formation decreased when 100 mM L-arginine hydrochloride was added compared to 50 mM. In addition, it was confirmed that the amount of aggregates and fragments increased according to the concentration of L-methionine and the presence or absence of D-mannitol was almost the same as or slightly increased compared to the basic buffer condition (Table 31 and Fig. 15).

[0186]

[0187] 3) Results of charge variant analysis

[0188] As a result of CEX-HPLC analysis, no significant change in charge variants was observed according to each excipient condition (Fig. 16).

[0189] 4) Viscosity and osmotic pressure analysis results

[0190] As a result of measuring viscosity and osmotic pressure according to each excipient concentration condition, it was confirmed that the viscosity reduction effect was greater under the condition of adding L-arginine hydrochloride at a concentration of 100 mM than 50 mM, and most of the osmotic pressure standards were satisfied at 112 to 588 mOsmol / kg (Table 32).

[0191]

[0192] Example 2.4.2. Secondary excipient concentration screening

[0193] In order to select the optimal concentration of L-arginine hydrochloride among the histidine basic buffer and two types of excipients selected in the primary excipient concentration screening of Example 1.4.1, two batches of HL161BKN samples (HL161BKN B018, HL161BKN B021) were tested for concentration, turbidity, purity (aggregates and fragments), viscosity, osmotic pressure, and stability 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, pH 6.0 was determined as the final formulation of HL161BKN (Table 33).

[0194]

[0195] 1) Concentration (A280) and turbidity (A340) analysis

[0196] The HL161BKN concentration showed little change over 4 weeks, which was expected to be due to the prevention of buffer evaporation by sealing the 1.5 mL microcentrifuge with parafilm and storing it in a constant temperature and humidity chamber (Table 34). In addition, turbidity showed a slight increase of less than 0.102 under all conditions (Table 35).

[0197]

[0198]

[0199] 2) Aggregate and fragment analysis

[0200] Through SEC-HPLC analysis, there was no significant difference in the amount of aggregates and fragments increased according to each excipient condition, but it was confirmed that the least amount of aggregates was generated in 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 Fig. 17).

[0201]

[0202] 3) Charge variant analysis

[0203] As a result of CEX-HPLC analysis, no significant change in charge variants (basic and acidic variants) was observed according to each excipient condition (Fig. 18).

[0204] 4) Viscosity and osmotic pressure analysis

[0205] As a result of viscosity and osmotic pressure measurements according to each excipient concentration condition, the viscosity was controlled to 11 to 16 cP, which is below the viscosity limit of 20 cp for subcutaneous administration products, and the osmotic pressure was 350 to 465 mOsmol / kg, which is suitable for the standard of 250 to 500 mOsmol / kg (Table 37).

[0206]

[0207] 5) Insoluble particulate analysis

[0208] Insoluble particulate analysis using microfluidic imaging (MFI) was performed on each excipient condition sample of HL161BKN at the Osong Advanced Medical Industry Promotion Foundation New Drug Development Support Center.

[0209] The samples of each condition were diluted to 10 mg / mL and the increase in the number of sub-visible particles ranging from 5 μm to 100 μm was compared. As a result, the increase in the number of sub-visible particles was the smallest in Condition 3 (Table 38 and Fig. 19).

[0210]

[0211] Example 3. Stability test

[0212] Long-term storage and accelerated stability tests for HL161BKN were conducted by Catalent (USA) for 36 months under the final formulation conditions. For stability assessment, the finished drug product (DP) was stored in borosilicate glass vials with Teflon-coated rubber stoppers. Stability was confirmed under the selected formulation conditions, confirming the stability of the DP for 36 months, confirming its potential for development as an injectable formulation (Tables 39 and 40).

[0213]

[0214]

[0215] Example 4. Selection of the final development candidate antibody formulation

[0216] Since HL161BKN shows a very stable tendency at high concentrations in the above formulation, we tried to confirm the possibility of developing it into a subcutaneous injection form. When developing an injection, it is known that a viscosity of 20 cP or less is suitable for subcutaneous administration to reduce pain and side effects at the injection site. Accordingly, HL161BKN was concentrated under 9 concentration conditions and the viscosity was measured at 5℃ and 25℃. The viscosity of a high-concentration HL161BKN sample of 170 mg / mL was confirmed to be 10 cP at 25℃, confirming that subcutaneous administration is possible (Fig. 20).

[0217] In the aforementioned formulation study, HL161BKN was confirmed to be highly stable even at high concentrations of 200 mg / mL or higher. This suggests potential for future development as a self-administered SC injection. Given that all competing products are infusion-based, this product offers potential differentiation through increased patient convenience.

[0218] In addition, since it was confirmed that high concentrations of HL161BKN are stable in the above formulation, it is expected that low concentrations of HL161BKN will also be stable in the above formulation. Therefore, the above formulation can be applied to HL161BKN at various concentrations.

Claims

1. (a) anti-FcRn antibody or fragment thereof; (b) one or more additives selected from mannitol, sorbitol, arginine, histidine, glycine and salts thereof; (c) a buffer system selected from citrate or histidine, and (d) Surfactant A pharmaceutical preparation having a pH of 4.0 to 8.0 containing:

2. In paragraph 1, The above additive is arginine or a salt thereof, A pharmaceutical preparation wherein the buffer system is histidine.

3. In paragraph 1, A pharmaceutical preparation wherein the surfactant is polysorbate.

4. In paragraph 1, A pharmaceutical preparation additionally comprising methionine.

5. In paragraph 1, A pharmaceutical preparation, characterized in that the above pharmaceutical preparation is in an injectable form.

6. In paragraph 1, A pharmaceutical preparation, characterized in that the pharmaceutical preparation has a viscosity of 20 cP or less.

7. In paragraph 1, A pharmaceutical preparation, characterized in that the pharmaceutical preparation has an osmotic pressure of 250 mOs / kg to 500 mOs / kg.

8. In paragraph 1, A pharmaceutical preparation wherein the concentration of the anti-FcRn antibody or fragment thereof is 50 mg / mL to 250 mg / mL.

9. In paragraph 8, A pharmaceutical preparation wherein the concentration of the anti-FcRn antibody or fragment thereof is 80 mg / mL to 250 mg / mL.

10. In paragraph 1, A pharmaceutical preparation, wherein the pH of the pharmaceutical preparation is 4.0 to 7.

0.

11. In paragraph 1, The above anti-FcRn antibody A heavy chain variable region comprising an H-CDR1 having an amino acid sequence of SEQ ID NO: 5, an H-CDR2 having an amino acid sequence of SEQ ID NO: 6, and an H-CDR3 having an amino acid sequence of SEQ ID NO: 7; A light chain variable region comprising an L-CDR1 having an amino acid of sequence number 8, an L-CDR2 having an amino acid of sequence number 9, and an L-CDR3 having an amino acid of sequence number 10. Pharmaceutical preparations.

12. In paragraph 1, A pharmaceutical preparation, characterized in that the pharmaceutical preparation is administered subcutaneously.

13. In paragraph 1, The above pharmaceutical preparation (a) 50 mg / mL to 250 mg / mL of anti-FcRn antibody, (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; A pharmaceutical preparation characterized by having a pH of 4.0 to 7.

0.

14. In paragraph 1, The above pharmaceutical formulation is a pharmaceutical formulation with increased stability, wherein the amount of aggregates and fragments of HL161BKN is 10% or less when stored for 6 months under accelerated conditions (25°C, relative humidity 60%).

15. In paragraph 1, The above pharmaceutical preparation is a pharmaceutical preparation with increased stability, wherein the amount of aggregates and fragments of HL161BKN is 10% or less when stored for 36 months under long-term storage conditions (5°C).

16. In paragraph 1, The above pharmaceutical preparation is a pharmaceutical preparation with increased stability, wherein the amount of aggregates and fragments of HL161BKN is 5.0% or less when stored for 36 months under long-term storage conditions (5°C).

17. In any one of paragraphs 1 to 16, The pharmaceutical preparation above is for the treatment of an autoimmune disease selected from the group consisting of myasthenia gravis, thyroid ophthalmopathy, global autoimmune hemolytic anemia, neuromyelitis optica, idiopathic thrombocytopenic purpura, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy, lupus nephritis, and membranous nephropathy.

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