Highly concentrated, highly stable antibody preparation and method for its production

Abstract

The present invention relates to antibody preparations. A high-concentration, high-stability antibody preparation and a method for its preparation are disclosed. The antibody preparation comprises an anti-C5 antibody with a light chain sequence of SEQ ID NO. 1 and a heavy chain sequence of SEQ ID NO. 2, or a modified, long-acting sequence thereof; and further comprises a buffer system consisting of: 20-50 mM histidine hydrochloride, 70-110 mM arginine, 25-50 mM sodium chloride, and 0.03% PS80. The concentration of a conventional antibody preparation is generally 100 mg / mL or less, and the antibody concentration of the buffer system reaches approximately 200 mg / mL. Furthermore, the antibody preparation exhibits good long-term stability, affinity, and bioavailability.

Description

Technical field

[0001] The invention belongs to the technical field of antibody production and relates in particular to an antibody preparation with high concentration and high stability, as well as a manufacturing process for it. State of the art

[0002] When developing therapies with biological antibodies for intravenous or subcutaneous administration at high concentrations, it is necessary to significantly concentrate the active ingredients (from approximately 20 mg / mL to approximately 100–150 mg / mL, or even 200 mg / mL or higher). For the treatment of chronic diseases requiring the administration of large single doses, such as PNH (paroxysmal nocturnal hemoglobinuria, which manifests as chronic intravascular hemolysis), antibody preparations with small dosage volumes and high concentrations must be developed to enable patients to self-medicate, thereby simplifying drug intake and increasing treatment adherence.However, the production of antibody drugs at high concentrations can lead to problems such as increased viscosity, protein aggregation, and changes in charge heterogeneity, potentially resulting in risks like reduced antibody activity and increased immunogenicity. Therefore, it is necessary to improve antibody drug formulations to increase antibody solubility, reduce viscosity, and address issues like protein aggregation and changes in charge heterogeneity.

[0003] Improvements to the formulation of the antibody drug essentially include the following aspects: I. Antibodies in the formulation; II. Calcium and acetate or buffer solution: The viscosity of the antibody preparations is reduced by adding calcium salts and / or acetates (and / or buffer solutions) to the preparations: The viscosity of the protein preparations is measured before and after the addition of calcium salts and / or acetates (and / or buffer solutions); III. Auxiliary substances in the formulation: Excipients can increase or improve the stability of protein formulations and, to some extent, influence the efficacy and immunogenicity of drugs. Therefore, it is necessary to develop protein formulations with stable and safe excipients. Excipients can include buffers, stabilizers, surfactants, etc.

[0004] The EA5 monoclonal antibody is a humanized monoclonal antibody specifically directed against the complement protein C5. Complement C5 can be enzymatically cleaved into C5a and C5b. C5a is an important pro-inflammatory molecule, while C5b can be further cleaved into C5b-9, the membrane attack complex (MAC), which leads to cell perforation or lysis. Deposition of the membrane attack complex on the surface of erythrocytes in patients with peripheral hypertension (PNH) can lead to erythrocyte lysis, the most important clinical manifestation of PNH. EA5 monoclonal antibodies can block the cleavage of C5 and prevent the formation of C5a and C5b-9. Therefore, they can be used for the effective treatment of PNH. Furthermore, they also have good therapeutic effects in diseases caused by terminal complement activation, such as aHUS and gMG.

[0005] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening clonal disorder of hematopoietic stem cells with an incidence of approximately 13 per million. Key clinical manifestations include hemolytic anemia, hemoglobinuria, venous thrombosis, bone marrow failure, and smooth muscle dysfunction. Intravascular hemolysis, caused by the complement system and targeting erythrocytes, is a central factor in the pathogenesis and death of PNH. CD59 is the primary inhibitor of the complement-membrane attack complex (MAC) on the surface of erythrocytes. In PNH patients, CD59 cannot bind to the cell membrane of erythrocytes due to an acquired somatic mutation in the phosphatidylinositol polysaccharide (PIG-A) gene on the X chromosome (Xp22.1), as the synthesis of glycosylphosphatidylinositol (GPI) is disrupted, ultimately leading to the loss of its function in inhibiting complement pathway activation.The most important treatments for PNH include complement inhibitors, glucocorticoids, and allogeneic hematopoietic stem cell transplantation. In particular, the complement C5 inhibitors Soliris (eculizumab) and Ultomiris (ravulizumab) are the standard treatments for PNH, effectively controlling intravascular hemolysis, reducing thrombus formation, and improving long-term survival. However, these drugs are expensive and must be administered intravenously, requiring regular hospital visits for infusions and causing significant inconvenience. Therefore, developing safe and effective subcutaneous injections of complement C5 inhibitors for the treatment of PNH patients is of paramount importance.

[0006] PNH is a typical disease caused by complement activation. EA5 antibodies also possess significant therapeutic potential for other diseases caused by complement activation, such as... Examples include rheumatoid arthritis, antiphospholipid syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria (PNH), dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, oligoimmune vasculitis, bullous epidermolysis, recurrent spontaneous abortion, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, allergic purpura nephritis, systemic lupus erythematosus-associated VasculitisRheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous air embolism, restenosis after stent implantation, coronary rotation atherectomy and percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory sepsis, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, Degos disease or catastrophic antiphospholipid syndrome. Content of the invention

[0007] To solve the aforementioned problems, this invention provides an anti-C5 antibody and its formulation, wherein the molecular design of the antibody and the buffer system of the formulation are optimized with the aim of improving the overall stability of the antibody formulation.

[0008] In this invention, “PS80” refers to polysorbate-80, also known as Tween-80.

[0009] In this invention, “histidine-histidine hydrochloride” refers to a buffer solution of sodium histidine + histidine hydrochloride.

[0010] In one aspect, the present invention provides an anti-C5 antibody.

[0011] The anti-C5 antibody is a modified antibody in which the light chain sequence is SEQ ID NO. 1 and the heavy chain sequence is SEQ ID NO. 2 or their long-acting modified sequences. This antibody molecule improves the solubility and stability of the antibody by modifying the Fc region. This increases the isoelectric point of the antibody to prevent antibody loss in subsequent processes and increases the antibody's affinity for FcRn to achieve a longer half-life in vivo.

[0012] Preferably, the long-acting modified sequence of the heavy chain is SEQ ID NO.3.

[0013] In one aspect, the present invention provides an antibody formulation containing the aforementioned anti-C5 antibody.

[0014] The anti-C5 antibody content is 0-200 mg / L, preferably 160-200 mg / mL, and even more preferably 180 mg / mL.

[0015] The antibody preparation also includes a buffer system.

[0016] The buffer system comprises: 20-50 mM histidine hydrochloride, 70-110 mM arginine, 25-50 mM sodium chloride and 0.03% PS80.

[0017] The histidine hydrochloride content can be selected from the following: 20-30 mM, 30-40 mM, 40-50 mM, 25-35 mM, 25-45 mM.

[0018] The arginine content can be selected from the following: 70-80 mM, 80-90 mM, 90-100 mM, 100-110 mM, 70-90 mM, 80-100 mM, 90-110 mM, 100-110 mM.

[0019] The sodium chloride content can be selected from the following: 25-30 mM, 25-35 mM, 30-50 mM, 40-50 mM, 35-40 mM, 25-45 mM, 30-45 mM.

[0020] Preferably, the buffer system comprises the following: 20 mM histidine hydrochloride, 70-110 mM arginine, 25-50 mM sodium chloride and 0.03% PS80.

[0021] Preferably, the buffer system comprises the following: 20 mM histidine hydrochloride, 80 mM arginine, 25-50 mM sodium chloride and 0.03% PS80.

[0022] Preferably, the buffer system comprises the following: 20 mM histidine hydrochloride, 80 mM arginine, 50 mM sodium chloride and 0.03% PS80.

[0023] The pH value of the buffer system is between 6.0 and 6.5.

[0024] Preferably, the pH value of the buffer system is between 6.0 and 6.3.

[0025] The pH value of the buffer system is preferably 6.3.

[0026] This invention also provides a buffer system that is used in antibody formulations.

[0027] In one aspect, the present invention relates to the use of the aforementioned anti-C5 antibody or antibody preparation for the manufacture of a drug.

[0028] The indications for the drug are selected from: rheumatoid arthritis, antiphospholipid syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome (aHUS), typical hemolytic uremic syndrome, peripheral neuropathy (PNH), dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, oligoimmune vasculitis, bullous epidermolysis, recurrent spontaneous abortion, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, allergic purpura nephritis, systemic lupus erythematosus-associated vasculitis. Rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki syndrome, venous air embolism, restenosis after stent implantation,coronary rotational atherectomy and percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory sepsis, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, Degos disease, or catastrophic antiphospholipid syndrome.

[0029] The indications for the drug are preferably PNH.

[0030] In one aspect, the present invention relates to a pharmaceutical product containing the aforementioned anti-C5 antibody or antibody preparation.

[0031] The drug is preferably used to treat PNH.

[0032] Preferably, the drug is administered by injection.

[0033] In one aspect, the present invention relates to the use of the above-mentioned buffer system for the preservation of antibody drugs.

[0034] Preferably, the antibody drug is an anti-C5 antibody-related drug, such as the aforementioned anti-C5 antibody. Advantageous effects of the present invention:

[0035] Compared to existing technology, the present invention improves the stability of anti-C5 antibodies, enhances the associated buffer system of antibody preparations, solves the problems of high viscosity, aggregation, and charge heterogeneity changes in antibody preparations, and improves the stability of corresponding highly concentrated liquid antibody preparations. The concentration of a conventional antibody preparation is generally 100 mg / mL or less, and the antibody concentration of the buffer system of the present disclosure reaches approximately 200 mg / mL. Furthermore, the antibody preparation exhibits good long-term stability, affinity, and bioavailability. Brief description of the drawings Fig. 1 is the inhibitory activity of the EA5 antibody at different concentrations against complement-dependent cytotoxicity (CDC). Fig.Figure 2 shows the curves of intravenous and subcutaneous injection of the antibody drug EA5 at a dose of 30 mg / kg to ICR mice. Detailed description

[0036] The present invention is explained in more detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but merely to illustrate it. Unless otherwise stated, the experimental methods used in the following embodiments, for which no specific conditions are given in the embodiments, are generally carried out under conventional conditions. Unless otherwise stated, the materials, reagents, etc., used in the following embodiment are all commercially available. Example 1 A method for the production of anti-C5 antibody formulations(1) Production of antibodies

[0037] A highly stable anti-C5 antibody whose heavy chain sequence was SEQ ID NO. 2 and whose light chain sequence was SEQ ID NO. 1. The antibody was modified to have a longer duration of action and an extended half-life. The heavy chain sequence of the modified antibody was SEQ ID NO. 3 and the light chain sequence was SEQ ID NO. 1.

[0038] The antibody production method used in this example is an industry-standard procedure. An expression vector was constructed and transfected into the host cell CHO via stable transfection to generate a stable cell line with high expression. The seed cells were subjected to batch feeding culture to express anti-C5 antibody. After harvesting, the anti-C5 antibody was isolated by affinity chromatography and ion-exchange chromatography. (2) Preparation of the buffer solution

[0039] The buffer solution was prepared according to the specified formula and the pH value of the buffer solution was adjusted to 6.3. (3) Production of the antibody preparation

[0040] A high-concentration antibody preparation was made using the modified antibody from step (1) and the buffer solution from step (2) at a concentration of 180 mg / mL. The preparation process is as follows: The centrifuge speed was set to 3500 rpm. The protein was concentrated using ultrafiltration centrifuge tubes and transferred to the preparation, a total of 155 times, and the protein concentration was adjusted to the target concentration of 180 mg / mL. Subsequently, each prescription sample was filtered and dispensed into 2R penicillin vials and 5 mL storage bags in the cleanroom workbench, and the penicillin vials were sealed. Recipe 1 Buffer: 50 mM histidine hydrochloride, 80 mM arginine hydrochloride, 25 mM sodium chloride, 0.03% (w / v) PS80. The pH of the buffer solution was adjusted to 6.3. Recipe 2 Buffer: 20 mM histidine hydrochloride, 80 mM arginine hydrochloride, 25 mM sodium chloride, 0.03% (w / v) PS80. Recipe 3 Buffer: 20 mM histidine hydrochloride, 110 mM arginine hydrochloride, 25 mM sodium chloride, 0.03% (w / v) PS80. Recipe 4 buffer: 20 mM citric acid sodium citrate + 70 mM arginine hydrochloride + 50 mM sodium chloride + 0.03% PS80 pH 6.3. Experimental Example 1: Study on the quality characteristics of antibody preparations

[0041] Recipe 1 was used as an example. The antibody preparation was tested for appearance, protein concentration, osmotic pressure, viscosity, SEC-HPLC, CEX-HPLC, nrCE-SDS, binding activity, affinity for C5, and CDC effect. (1) Test method for determining protein concentration: The protein content was determined using an ultraviolet spectrophotometer (Shimadzu, model: UV-1900). The extinction coefficient of the protein was 1.531 (mg / mL). -1 ·cm -1 The microvolume cuvettes were rinsed 2-3 times with ultrapure water. Subsequently, 200 µL of ultrapure water per vessel was added to each of the two microvolume cuvettes. A blank correction was performed, and then the sample (200 µL per vessel) was added. The results were read three times consecutively. The sample concentration was calculated using the extinction coefficient, the dilution factor, and the OD value. (2) The procedure for measuring the osmotic pressure was as follows: The sample was placed on the probe of the osmometer. The instrument started the measurement automatically, and the measurement results were displayed and printed after 1 minute. (3) The viscosity test was performed as follows: A specific sample volume was taken using a micro-VISC pipette syringe. The syringe needle was pressed against the injection port. After the sample was inserted, the instrument started the measurement automatically. (4) Manufacturer of the SEC-HPLC detection column: Agilent, model AdvanceBio SEC 300A, specification 2.7µm.

[0042] Manufacturers of high-performance liquid chromatographs (HPLC): Thermo, UltiMate 3000.

[0043] The mobile phase consisted of 20 mM PB + 200 mM NaCl + 100 mM Arg with a pH of 6.8.

[0044] The main peak, the polymer peak, and the fragment peak after monomer were calculated by integration using the surface normalization method.

[0045] The specific conditions for the SEC-HPLC purity test were as follows: Chromatographic conditions Chromatographic parameters Detector wavelength 280 nm Column temperature 25°C flow rate 0.5 ml / min Sample injection volume 100µg Duration 30 min (5) CEX-HPLC purity

[0046] Chromatographic column: Sepax, model Proteomix SCX-NP5, size 4.6 × 250 mm; High-performance liquid chromatograph (HPLC): Thermo, model UltiMate 3000. Mobile phase A: 20 mM MES, pH 6.5; Mobile phase B: 20 ​​mM MES + 500 mM NaCl, pH 6.5. Peak area normalization was used for quantitative analysis of the results. The concentrations of acidic components (sum of the percentage area of ​​all peaks before the main peak), main components (percentage area of ​​the main peak), and alkaline components (sum of the percentage area of ​​all peaks after the main peak) were calculated. Details are given in the following table: CEX-HPLC purity testing method Chromatographic conditions Chromatographic parameters Detection wavelength 280 nm Column oven 30°C Temperature of the sample container 10°C Sample injection volume 50µL Collection time 30 min Gradient elution program Time Mobile Phase A (%) Mobile Phase B (%) 0,00 100,0 0,0 5,00 100,0 0,0 20,00 70,0 30,0 20,01 0,0 100,0 25,00 0,0 100,0 25,01 100,0 0,0 30,00 100,0 0,0 (6) nrCE-SDS purity

[0047] Capillary electrophoresis system: Manufacturer SCIEX, model PA800 Plus. Capillary electrophoresis chromatograph: Manufacturer G7100A, model Agilent. The sample was first desalted, then citrate-phosphate buffer with a pH of 6.5 was added and concentrated to 400 µL by centrifugation. The concentrated column was centrifuged for 15 minutes at 13,000 rpm, and then transferred to a new centrifuge tube and centrifuged for 3 minutes at 6,000 rpm to obtain the protein. A 250 mM NEM solution was added, thoroughly mixed, heated to 70°C for 10 minutes, cooled, and centrifuged for 3 minutes at 10,000 rpm before injection. A PDA detector with a detection window of 100 × 800 µm was used. Samples were injected from the left end of the capillary. The effective separation length of the capillary was 20 cm. Voltage injection with a duration of 20 seconds at 5 kV was used.A constant voltage separation of 15 kV was used. Rinse for 3 minutes at 70 psi with alkaline wash buffer, 1 minute at 70 psi with acidic wash buffer, 1 minute at 70 psi with pure water, and 10 minutes at 70 psi with SDS gel buffer. The content of polymer components (the sum of the percentage area fractions of all peaks before the main peak), major components (the percentage area fraction of the main peak), and low molecular weight components (the sum of the percentage area fractions of all peaks after the main peak) was calculated using the peak area normalization method. (7) Methods for determining binding activity (ELISA):

[0048] 250 µg / mL of C5 antigen (obtained from Acro Bio, catalog number COS-H52Ha) was diluted to 0.5 µg / mL with 1×PBS coating buffer. The 96-well plate was then coated with the aforementioned diluted solution at a rate of 100 µL / well at 4 °C overnight. The plate was washed four times, blocking solution was added at a rate of 250 µL / well, and the plate was incubated for two hours at room temperature. The plate was washed four times, and the gradient-diluted samples were transferred to the 96-well ELISA plate at a rate of 100 µL / well and incubated for one hour at room temperature. After four further washes, the secondary antibody working solution (diluted 1:20,000) was added at a volume of 100 µL / well and incubated for 1 hour at room temperature. The plate was then washed four more times, TMB staining reaction solution was added at 100 µL / well, and incubated for 10 minutes at room temperature. Finally, the stop solution was added.The absorbance value at 450 nm was measured using an ELISA reader, with 630 nm serving as the reference wavelength. A four-parameter curve fit was performed, with concentration on the x-axis and OD on the x-axis. 450-630nm performed on the y-axis. EC 50 and R 2 The relative binding activity of the sample to be tested could be determined directly, and it was calculated. Relative binding activity = EC50 of the reference (or working reference) / EC50 of the sample to be tested × 100%.

[0049] The most important quality characteristics to be checked were the following: Test items method Standard range Modified antibodies 2. Purity and impurities purity SEC-HPLC method Monomer: ≥90.0% 99,0% Polymer content: ≤5.0% 1,0% Non-reducing CE-SDS method Main peak: ≥90.0% 96,4 Reduced CE-SDS method Main peak: ≥90.0% 99,2 Charging option CEX-HPLC method Acid components (%): Reported data 12,3 Peak (%): Reported data 72,8 Alkaline components (%): Reported data 14,9 3. Valence Binding activity ELISA method 70-130% 81% 4. Contents Protein content UV method 162.0-198.0 mg / mL 181,0 5. Others Look Observation method Clear, slightly yellowish liquid Clear, slightly yellowish liquid viscosity Viscometer (cP) <15 12,64 Osmotic pressure Osmotic pressure at freezing point (mOsmol / kg) 280-310 290 Experimental Example 2: Affinity test of antibody preparations

[0050] (1) The affinity between the anti-C5 antibody and C5 was tested using the following procedure: The biotin-labeled C5 was diluted to 5 µg / mL with SD buffer (0.1% BSA, 0.01% Tween 20, 1×PBS), and the test sample was diluted to 50 µg / mL. Three replicates were prepared in parallel. The diluted test sample, FcRn, and SD buffer were each pipetted into black 96-well plates. The SA sensor and black 96-well plates were inserted into the instrument, and the program was configured for curve fitting and data analysis. The test results for the preparation of formulation 1, as an example, are presented as follows: koff (1 / s) con (1 / s) KD (M) React Recipe 1 4,45E-05 1,95E+04 2.28E-09 1,37

[0051] The results showed that the anti-C5 antibody of the present invention has a good affinity for C5.

[0052] (2) The affinity between the antibody and FcRn was tested using the following procedure: Prior to the experiment, the pH of the SD buffer was adjusted to 6.0. The biotin-labeled FcRn was diluted to 5 µg / mL with SD buffer, and the test sample was diluted to 50 µg / mL. Three replicates were prepared in parallel. The diluted test sample, FcRn, and buffer were each pipetted into black 96-well plates. The SA sensor and black 96-well plates were inserted into the instrument, and the program was configured for curve fitting and data analysis. Eculizumab (see US 6355245; the expression plasmid was constructed. Transient transfection and expression were performed using 293T cells with PEIUS5736137A. After further affinity chromatography, buffer solution was added and stored until use) was used as a positive control.

[0053] The test results were as follows: koff (1 / s) con (1 / s) KD (M) React Recipe 1 0,000664 5.82E+05 1.14E-09 0,776 Control substance 0,00758 7.08E+05 1.07E-08 0,301

[0054] The test results show that the antibody in the antibody preparation of the present invention has a better binding capacity to FcRn than the positive control.

[0055] (3) Testing the CDC effect at different antibody concentrations. The test procedure is as follows: A suitable number of target cells (human Burkitt lymphoma cells, ATCC CCL-213) were taken and 10 µg / mL of rituximab (see US5736137A; the expression plasmid was constructed, and transient transfection and expression were performed using 293T cells with PEI. After further affinity chromatography, buffer solution was added and stored until use) was added and incubated for half an hour. The antibody drug was then added at a concentration of 216 ng / mL to 12500 ng / mL and incubated for 3 hours, removed, and brought to room temperature. The reagent for the cell titer test (Promega, catalog number G7572) was then added, the mixture was shaken in the dark, and the value was read. A curve fitting was performed using four parameters, with antibody drug concentration plotted on the x-axis and RLU value on the y-axis. The RLU value correlated positively with the cell lysis rate.The result is in . Fig. 1 shown. Fig. Figure 1 shows that as antibody concentration decreases, the cell lysis rate increases and the complement-induced CDC effect caused by rituximab is enhanced, while high concentrations of anti-C5 antibodies can inhibit the CDC effect caused by rituximab. Experimental Example 3: Accelerated Stability Experiment

[0056] The antibody formulations of recipes 1-4 were stored at 40°C for 14 days. To assess the stability of the formulations, their initial parameters (T0) and the parameters after 14 days were measured as in Experimental Example 1. The results were as follows: Recipe 1 Recipe 2 Recipe 3 Recipe 4 0 days 14 days 0 days 14 days 0 days 14 days 0 days 14 days pH 6,40 6,40 6,40 6,28 6,40 6,40 6,39 6,28 Protein concentration (mg / mL) 180,5 179,9 181,9 182,2 179,4 179,6 185,1 183,6 Look Clear, slightly yellowish liquid Clear, slightly yellowish liquid Clear, colorless liquid Clear, colorless liquid Clear, slightly yellowish liquid Clear, slightly yellowish liquid Clear, colorless liquid Clear, colorless liquid SEC-HPLC_Monomer% 98,3 95,1 98,9 94,0 98,3 94,3 99,0 96,9 SEC-HPLC_HM W% 1,4 3,7 0,8 1,5 1,5 4,5 0,8 1,7 SEC-HPLC_LM W% 0,3 1,3 0,3 4,5 0,3 1,3 0,1 1,4 nrCE-SDS_Haupt peak % 96,8 93,5 98,2 95,3 96,6 92,9 98,4 97,5 nrCE-SDS_LMW % 3,2 5,8 1,8 4,7 3,4 5,8 1,6 2,5 nrCE-SDS_HM W% 0,0 0,8 0,0 0,0 0,0 1,3 0,0 0,0 CEX-HPLC_Säu repeak % 22,7 30,3 23,0 32,3 22,7 29,5 22,9 31,0 CEX-HPLC_Hau ptpeak % 64,5 52,9 61,9 58,8 64,4 53,1 62,0 51,3 CEX-HPLC_Alk alipeak % 12,8 16,8 15,2 18,9 12,9 17,4 15,1 17,8 Experimental Example 4: Bioavailability of subcutaneous injection

[0057] Formulation 1 was used as an example in the bioavailability study of the antibody preparation. The detailed procedure is as follows: In this experiment, CD-1 mice were injected with an antibody preparation subcutaneously and intravenously. Subsequently, changes in antibody concentration in the mice were observed over time, and the basic pharmacokinetic parameters of the drug were determined to understand the fundamental kinetic properties of the tested substances.

[0058] The specific procedure is as follows: ICR mice (8 weeks old, 30g) randomly assigned by weight to groups of 6 animals each (half male and half female) received a single intravenous injection of the antibody preparation into the tail vein or a single subcutaneous injection of the antibody preparation into the neck at a dose of 10 mL / kg.

[0059] Sampling time: immediately after administration (within 1 minute), 1 hour ± 5 minutes, 6 hours ± 5 minutes, 24 hours ± 5 minutes, 48 ​​hours ± 5 minutes, 72 hours ± 5 minutes, 96 hours ± 5 minutes, 120 hours ± 5 minutes, 168 hours ± 5 minutes, 240 hours ± 5 minutes, 336 hours ± 5 minutes, 504 hours ± 5 minutes, and 672 hours ± 5 minutes after administration; Blood volume: approximately 0.2 mL / animal / time point; Number of animals sampled: 3 animals / time point / sex / group; Blood collection method: vein at the eye socket. The concentration of antibodies in the plasma samples was analyzed using a methodologically validated ELISA test. All samples were measured in duplicate. The concentration and time profiles of antibodies in plasma were analyzed using a non-compartmental model.

[0060] ELISA method for determining antibody levels in plasma: A polyclonal goat anti-human antibody (obtained from Abcam, catalog number Ab6858) was applied to a hydrophobic ELISA plate, and the plasma EA5 content was determined using the double antibody sandwich method. The results are presented in Fig. 2 shown. Fig. Figure 2 shows that subcutaneous injection, compared to intravenous injection, is mainly absorbed via the lymphatic system, with the maximum drug concentration being reached after 24 hours, with an average Cmax of 240 µg / mL, and with a bioavailability of approximately 70%. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] US 6355245

[0052] US 5736137A

[0055]

Claims

An antibody preparation comprising an anti-C5 antibody and a buffer system, wherein a sequence of a light chain of the anti-C5 antibody is SEQ ID NO. 1, and a sequence of a heavy chain of the anti-C5 antibody is SEQ ID NO. 3; the buffer system comprises 20-50 mM histidine hydrochloride, 70-110 mM arginine, 25-50 mM sodium chloride and 0.03% PS80; the anti-C5 antibody concentration is 160-200 mg / mL. The antibody preparation according to claim 1, characterized in that the buffer system comprises 20 mM histidine hydrochloride, 70-110 mM arginine, 25-50 mM sodium chloride and 0.03% PS80. The antibody preparation according to claim 2, characterized in that the buffer system comprises 20 mM histidine hydrochloride, 80 mM arginine, 25-50 mM sodium chloride and 0.03% PS80. The antibody preparation according to claim 3, characterized in that the buffer system comprises 20 mM histidine hydrochloride, 80 mM arginine, 50 mM sodium chloride and 0.03% PS80. The antibody preparation according to claim 4, characterized in that the pH value of the buffer system is 6.0-6.

5. The antibody preparation according to claim 5, characterized in that the pH value of the buffer system is 6.0-6.

3. The antibody preparation according to any one of claims 1-6 for use in the manufacture of a medicament, characterized in that the medicament is used for the treatment of one or more of the following diseases: rheumatoid arthritis, antiphospholipid syndrome, lupus nephritis, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, typical hemolytic uremic syndrome, peripheral neuropathy (PNH), dense deposit disease, neuromyelitis optica, multifocal motor neuropathy, multiple sclerosis, macular degeneration, HELLP syndrome, spontaneous abortion, thrombotic thrombocytopenic purpura, oligoimmune vasculitis, bullous epidermolysis, recurrent spontaneous abortion, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, allergic Purpuric nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis,Immune complex vasculitis, Takayasu disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous air embolism, restenosis after stent implantation, coronary rotation atherectomy and percutaneous transluminal coronary angioplasty, myasthenia gravis, cold agglutinin disease, dermatomyositis, paroxysmal cold hemoglobinuria, antiphospholipid syndrome, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory sepsis, septic shock, spinal cord injury, glomerulonephritis, transplant rejection, Hashimoto's thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, Degos disease, or catastrophic antiphospholipid syndrome. The antibody preparation according to claim 7, characterized in that the medicinal product is a subcutaneous injectable medicinal product. A subcutaneous injectable drug comprising the antibody preparation according to any one of claims 1-6.

Images