High concentration anti-c5 antibody formulations

Abstract

The present disclosure relates to stable aqueous solutions comprising high concentrations of an anti-C5 antibody (e.g., ravulizumab) and methods of making the solutions. The present disclosure also provides methods of using the solutions to treat or prevent complement-associated disorders, such as paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). Therapeutic kits containing one or more of the solutions, and means for administering the solutions to a patient in need of such treatment, are also contemplated.

Description

[0001] This application is a divisional application of the invention patent application filed on July 27, 2018, with application number 201880048000.5 and invention title "High Concentration Anti-C5 Antibody Preparation".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 537,741, filed July 27, 2017. The entire contents of the aforementioned provisional patent application are incorporated herein by reference.

[0004] sequence list

[0005] This application contains a sequence list, which has been submitted electronically in ASCII format and incorporated herein by reference in its entirety. The ASCII copy, created on July 26, 2018, is named AXJ-226PC_SL.txt and is 32,987 bytes in size. Background Technology

[0006] The complement system works in conjunction with other immune systems to defend against invasion by cellular and viral pathogens. At least 25 complement proteins exist, found as a complex collection of plasma proteins and membrane cofactors. Plasma proteins account for approximately 10% of vertebrate serum globulins. The complement components exert their immune defense functions through interactions in a complex but precise series of enzymatic cleavage and membrane-binding events. The resulting complement cascade leads to the production of products with opsonization, immunomodulatory, and cleavage functions. For example, a concise summary of the biological activities associated with complement activation is provided in The Merck Manual, 16th edition.

[0007] While a properly functioning complement system provides robust defense against infectious microorganisms, inappropriate regulation or activation of the complement pathway has been involved in the pathogenesis of a variety of conditions, including paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) (see, for example, Socié G et al., French Society of Haematology. Lancet. 1996; 348(9027): 573-577; Brodsky, R., Blood. 2014; 124(18): 2804-2811); Hillmen, P. et al., Am. J. Hematol. 2010; 85(8): 553-559; Caprioli et al. (2006) Blood 108 : 1267-1279; and Kavanagh et al. (2006) British Medical Bulletin 77 and 78: 5-22).

[0008] Patients with complement-related disorders (such as PNH or aHUS) are at high risk of morbidity and death. Therefore, the object of the present invention is to provide improved compositions and methods for treating patients with complement-related disorders. Summary of the Invention

[0009] This document provides stable, highly concentrated aqueous solutions of anti-C5 antibodies, as well as methods for preparing and using said formulations. Among other things, this disclosure provides formulation conditions suitable for maintaining the physical and functional stability of anti-C5 antibodies (e.g., ravulizumab, also known as "antibody BNJ441" and "ALXN1210") in high-concentration solutions for a considerable period of time. For example, this disclosure provides formulation conditions capable of maintaining anti-C5 antibodies in predominantly monomeric form for up to 2 years at 2°C to 8°C, even when the antibody is maintained in solution at a concentration of approximately 100 mg / mL or higher. Furthermore, as described herein and illustrated in the working examples, such formulations also minimize the aggregation, fragmentation, or degradation of anti-C5 antibodies (e.g., ravulizumab) in highly concentrated solutions. For example, this disclosure provides preparation conditions that enable the anti-C5 antibody to remain in a highly concentrated form for two years without detectable antibody fragmentation or degradation products (such as those determined using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), such as HPLC-gel permeation) and with aggregates not exceeding 2%. This document also provides conditions suitable for preparing solutions of anti-C5 antibodies (such as revolucratab) at concentrations higher than 200 mg / mL.

[0010] The benefits of stable, highly concentrated aqueous solutions of anti-C5 antibodies are numerous. First, for therapeutic applications requiring administration of antibodies to patients in small volumes, efficacy often depends on the amount of antibody that can be administered within that small volume. In cases where high concentrations of anti-C5 antibodies cannot be formulated, delivery routes such as subcutaneous, intravitreal, and / or intra-articular administration are often avoided. Accordingly, highly concentrated antibody formulations offer patients more options regarding the route of administration. For therapeutic applications requiring frequent, long-term administration and / or self-delivery, high-concentration formulations make administration possible and are more appealing to patients than intravenous infusion. For example, high-concentration formulations of anti-C5 antibodies allow patients to self-administer antibodies via, for example, subcutaneous or intravenous injection. Therefore, the ability to formulate high-concentration antibodies can improve administration adherence by providing a convenient home administration alternative for patients with complement-related conditions.

[0011] Furthermore, the method for generating the aqueous solution described herein does not require a lyophilization step, and the high-concentration aqueous solution specifically reported does not require reconstitution from lyophilized material. The currently reported high-concentration antibody solution offers several advantages over reconstituted lyophilized antibody formulations. First, physicians must reconstitute the lyophilized antibody solution locally under aseptic conditions, which increases the chance of microbial contamination of the solution before administration. Additionally, reconstitution requires considerable care to ensure that all solids contained in the reconstitution container are properly dissolved in the solution. Therefore, the high-concentration aqueous solution provided herein offers physicians, nurses, and / or patients a rapid, easy, safe, and effective way to deliver therapeutic antibodies to patients in need.

[0012] Other benefits of high-concentration formulations include, for example, manufacturing cost savings due to reduced storage space and / or product fill counts. Additionally, the ability to produce products with longer shelf lives will ultimately require fewer production runs, which ultimately lowers costs for both manufacturers and consumers of highly concentrated therapeutic antibodies.

[0013] An exemplary anti-C5 antibody is revoizumab (also known as antibodies BNJ441 and ALXN1210), comprising heavy and light chains having the sequences shown in SEQ ID NO: 14 and 11, respectively, or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises complementarity-determining regions (CDRs) or variable regions (VRs) of the heavy and light chains of revoizumab. Thus, in one embodiment, the antibody comprises CDR1, CDR2, and CDR3 domains of the revoizumab heavy chain variable (VH) region having the sequence shown in SEQ ID NO: 12, and CDR1, CDR2, and CDR3 domains of the revoizumab light chain variable (VL) region having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises CDR1, CDR2, and CDR3 heavy chain sequences as shown in SEQ ID NO: 19, 18, and 3, respectively, and CDR1, CDR2, and CDR3 light chain sequences as shown in SEQ ID NO: 4, 5, and 6, respectively.

[0014] In another embodiment, the antibody comprises VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0015] In another embodiment, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 13.

[0016] In another embodiment, the antibody comprises a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region contains Met-429-Leu and Asn-435-Ser substitutions at methionine 428 and asparagine 434 residues corresponding to the natural human IgG Fc constant region, respectively, in accordance with EU designations.

[0017] In another embodiment, the antibody comprises heavy chain sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 19, 18, and 3, respectively, and light chain sequences of CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 4, 5, and 6, respectively, and a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region contains Met-429-Leu and Asn-435-Ser substitutions at methionine 428 and asparagine 434 residues corresponding to the natural human IgG Fc constant region, respectively, according to EU numbers.

[0018] In another embodiment, the antibody competes with the antibodies mentioned above for binding to and / or binding to the same epitope on C5. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibodies mentioned above (e.g., at least about 90%, 95%, or 99% variable region identity with SEQ ID NO: 12 and SEQ ID NO: 8).

[0019] In another embodiment, the antibody binds to human C5 at pH 7.4 and 25°C, with an affinity dissociation constant (K0). D In 0.1nM≤K D Within the range of ≤1 nM. In another embodiment, the antibody binds to human C5 at pH 6.0 and 25°C, K D ≥10 nM. In another embodiment, the antibody [(the antibody or its antigen-binding fragment is effective against human C5 K at pH 6.0 and 25°C]. D The antibody or its antigen-binding fragment is effective against human C5 antigen at pH 7.4 and 25°C. D )] greater than 25.

[0020] On one hand, a stable aqueous solution (e.g., a sterile solution) is provided, wherein the solution contains an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6. In another embodiment, the solution contains an anti-C5 antibody (e.g., revozumab) at a concentration of 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 mg / mL.

[0021] In another embodiment, the stabilized aqueous solution comprises one or more additional agents (e.g., stabilizers, buffers, surfactants, and / or preservatives). For example, in one embodiment, the stabilized aqueous solution comprises a stabilizer. Exemplary stabilizers include, but are not limited to, polyols, sugars (e.g., sucrose or trehalose), amino acids (e.g., arginine), amines, and salting-out salts. In one embodiment, the solution comprises at least one stabilizer at a concentration of 2-10% (inclusive). In one embodiment, the solution comprises 5% sucrose. In another embodiment, the solution comprises at least one or more stabilizers at a concentration of 10 mM to 50 mM (inclusive). In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution comprises 25 mM arginine.

[0022] In another embodiment, the solution contains at least one or more buffers. Non-limiting examples of typical buffers that may be included in the washing solution include Tris (tris(hydroxymethyl)methylamine), bis-Tris, bis-Trispropane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N-morpholine)ethanesulfonic acid), phosphate, HEPES (4-2-hydroxyethyl-1-piperazine ethanesulfonic acid), citrate, MOPS (3-(N-morpholine)propanesulfonic acid), TAPS (3{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N-tris( The buffer comprises (hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsine salt (dimethylarsine), SSC (sodium citrate saline), and sodium phosphate. In another embodiment, the buffer is an amino acid. The amino acid may be, for example, selected from histidine (e.g., L-histidine), serine (e.g., L-serine), and glycine (e.g., L-glycine). In another embodiment, the solution contains two or more buffers. In one particular embodiment, the buffer is sodium phosphate.

[0023] In another embodiment, the solution contains at least one or more buffers with a concentration of 10 mM to 300 mM (inclusive). In another embodiment, the solution contains at least one buffer with a concentration of 10 mM to 200 mM (inclusive). In another embodiment, the solution contains at least one buffer with a concentration of 10 mM to 100 mM (inclusive). In another embodiment, the solution contains at least one buffer with a concentration of 10 mM to 50 mM (inclusive). In another embodiment, the solution contains at least one buffer with a concentration of 20 mM to 50 mM (inclusive). In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 20 mM. In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 50 mM.

[0024] In another embodiment, the solution contains a carbohydrate excipient at a concentration of 0.1% to 5%. In one embodiment, the carbohydrate excipient is present in the solution at a concentration of at least or equal to 1.5%. In another embodiment, the carbohydrate excipient is present in the solution at a concentration of at least or equal to 3%. The carbohydrate excipient may be, for example, a carbohydrate excipient selected from sorbitol and mannitol. In another embodiment, the solution contains two or more carbohydrate excipients.

[0025] In another embodiment, the solution comprises a surfactant. Surfactants suitable for use in the formulations of the present invention include, but are not limited to, fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, fatty acid glycerides (e.g., glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate), fatty acid polyglycerides (e.g., glyceryl monostearate, glyceryl distearate, glyceryl monolinoleate), and polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate). Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene fatty acid glycerides (e.g., polyoxyethylene monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., Polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitan beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene...). Ethylene stearamide; C12-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (with an average addition of 2 to 4 moles of ethylene oxide units) (e.g., sodium polyoxyethylene lauryl sulfate) and C10-C18 alkyl sulfonate succinates (e.g., sodium lauryl sulfonate succinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids.

[0026] In one embodiment, the surfactant in the formulation is a nonionic surfactant. In some embodiments, the surfactant in the formulation is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more thereof. In one embodiment, the surfactant in the formulation is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the formulation is polysorbate 60. In another embodiment, the surfactant in the formulation is polysorbate 40. In yet another embodiment, the surfactant in the formulation is polysorbate 20 (Tween 20). The concentration of the surfactant in the solution may, for example, be between 0.001% and 0.02% (inclusive). For example, the surfactant may be present in the formulation in an amount from about 0.001% to about 1%, or from about 0.001% to about 0.5%, or from about 0.01% to about 0.2%. In one embodiment, the aqueous solution contains at least or about 0.001% (e.g., at least or about 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%). Surfactants of 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, or 0.5% or higher. In another embodiment, the aqueous solution contains no more than 0.2% (e.g., no more than 0.19%, 0.18%, 0.17%, 0.16%, 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or 0.001%) of a pharmaceutically acceptable surfactant. In one particular embodiment, the surfactant is 0.05% polysorbate 80.

[0027] In another embodiment, the solution contains a preservative. Exemplary preservatives include, but are not limited to, benzyl alcohol, m-cresol, and phenol.

[0028] In one embodiment, the stable aqueous solution contains no more than five agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In yet another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

[0029] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50 ± 15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5 ± 3% (e.g., 2%, 3%, 4%, 5%) 6%, 7% or 8% sucrose; and 25 ± 10 mM (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM) arginine; wherein the pH of the solution is 7.4 ± 0.5 (e.g. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0030] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50 ± 15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5 ± 3% (e.g., 2%, 3%, 4%, 5%) 6%, 7% or 8% sucrose; and 25 ± 10 mM (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM) arginine; wherein the pH of the solution is 7.4 ± 0.5 (e.g. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0031] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50±15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5±3% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, or 8%) sucrose; 25±10 mM (e.g., 15, 16, 17, ... Arginine (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM); and 0.05 ± 0.03% (e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08%) of polysorbate 80, wherein the pH of the solution is 7.4 ± 0.5 (e.g., 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0032] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50±15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5±3% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, or 8%) sucrose; 25±10 mM (e.g., 15, 16, 17, ... Arginine (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM); and 0.05 ± 0.03% (e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08%) of polysorbate 80, wherein the pH of the solution is 7.4 ± 0.5 (e.g., 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0033] In another embodiment, a stable aqueous solution (e.g., a sterile solution) is provided, wherein the solution comprises (a) an anti-C5 antibody (e.g., reveluzumab), (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, a stable aqueous solution (e.g., a sterile solution) is provided, wherein the solution comprises (a) an anti-C5 antibody (e.g., reveluzumab) at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody (e.g., reveluzumab), (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine. In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody (e.g., revozumab) at a concentration of 100 mg / mL, (b) 50 mM phosphate buffer, (c) 5% sucrose, and (d) 25 mM arginine.

[0034] In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

[0035] In another embodiment, the stable aqueous solution comprises (a) anti-C5 antibody, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine. In another embodiment, the stable aqueous solution comprises (a) anti-C5 antibody at a concentration of 100 mg / mL, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine. In another embodiment, the stable aqueous solution comprises (a) anti-C5 antibody at a concentration of 100 mg / mL, (b) 50 mM phosphate buffer, (c) 5% sucrose, (d) 0.05% polysorbate 80, and (e) 25 mM arginine.

[0036] In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In yet another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

[0037] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine.

[0038] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine.

[0039] In one embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine.

[0040] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

[0041] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0042] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; (d) 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0043] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0044] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; (d) 0.05% polysorbate 80; and (e) 25 mM arginine.

[0045] In one embodiment, the pH is 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or 7.9. In another embodiment, the pH of the solution is between 7.0 and 7.4. In yet another embodiment, the pH of the solution is between 7.2 and 7.8. In yet another embodiment, the pH of the solution is between 7.2 and 7.6. In a particular embodiment, the pH of the solution is 7.4.

[0046] The solutions described herein can be formulated for any suitable mode of administration. In one embodiment, the solution is formulated for administration via a parenteral route (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection). In a particular embodiment, the solution is formulated for subcutaneous administration. For example, in one embodiment, the stable aqueous solution contains 100 mg / mL of anti-C5 antibody and is formulated for subcutaneous administration. In another particular embodiment, the solution is formulated for intravenous administration. For example, in one embodiment, the stable aqueous solution contains 100 mg / mL of anti-C5 antibody and is formulated for intravenous administration.

[0047] In one embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revolucratabumab) retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least nine months. In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least one year. In yet another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least 18 months. In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of its monomer content during storage at 2°C to 8°C for at least two years, as determined by SEC-HPLC.

[0048] In another embodiment of any of the solutions described herein, less than 5% of the anti-C5 antibody (e.g., revozumab) aggregates in the solution, as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, less than 4% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In another embodiment, less than 3% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In another embodiment, less than 2% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In another embodiment, less than 1% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC.

[0049] In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least six months. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least nine months. In another embodiment, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least one year. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least eighteen months. In another embodiment, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least two years. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least three years.

[0050] In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least nine months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least six months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least one year. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least 18 months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least two years.In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least three years.

[0051] On the other hand, a method is provided for generating a stable concentrated antibody solution, the solution comprising: 100 mg / mL anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose; and 25 mM arginine, the method comprising:

[0052] i) Provide a first aqueous solution comprising the anti-C5 antibody, the first aqueous solution having a first formulation and containing no more than 10 mg / mL of the anti-C5 antibody;

[0053] ii) Percolate the first aqueous solution into a formulation containing 50 mM phosphate buffer, 5% sucrose, and 25 mM arginine at pH 7.4 to produce a second aqueous solution, wherein the second aqueous solution has a second formulation due to percolation; and

[0054] iii) Concentrate the second aqueous solution to produce a stable concentrated antibody solution containing 100 mg / mL of the anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine.

[0055] In another embodiment, a method is provided for producing a stable concentrated antibody solution comprising 100 mg / mL anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, and 0.05% polysorbate 80, the method comprising:

[0056] i) Provide a first aqueous solution comprising the anti-C5 antibody, the first aqueous solution having a first formulation and containing no more than 10 mg / mL of the anti-C5 antibody;

[0057] ii) Percolate the first aqueous solution into a formulation containing 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, and 0.05% polysorbate 80 at pH 7.4, thereby producing a second aqueous solution, wherein the second aqueous solution has a second formulation due to percolation; and

[0058] iii) Concentrate the second aqueous solution to produce a stable concentrated antibody solution containing 100 mg / mL of the anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80.

[0059] Methods for treating human patients with complement-related conditions are also provided, comprising administering to the patient (e.g., subcutaneously or intravenously) a stable aqueous solution as described herein in an amount effective in treating complement-related conditions. Exemplary complement-related conditions include, but are not limited to, 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, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / enterovascular disease, vasculitis, and Henoch-Schonlein purpura nephritis. (e.g., nephritis), systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous air embolism, stent placement, coronary rotational atherosclerosis, restenosis after 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. Thyroiditis, type 1 diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture's syndrome, Degos disease, and catastrophic antiphospholipid syndrome. In one particular embodiment, the complement-related symptom is atypical hemolytic uremic syndrome (aHUS). In another embodiment, the complement-related symptom is paroxysmal nocturnal hemoglobinuria (PNH).

[0060] A kit is also provided comprising a stable aqueous solution, as described herein, suitable for use in the therapeutically effective amounts of the methods described herein. In one embodiment, the kit comprises: (i) any solution described herein; and (ii) a tool (e.g., a syringe) for delivering the solution to a patient in need. In one embodiment, the tool is adapted for subcutaneous delivery of the solution to a patient. In one embodiment, the tool is adapted for intravenous delivery of the solution to a patient. In another embodiment, the kit further comprises at least one additional active agent for treating complement-related conditions in a subject. Attached Figure Description

[0061] Figure 1 Dynamic light scattering results were depicted for salt titration of 50 mg / mL histidine-buffered saline-exchanged revozumab (ALXN1210).

[0062] Figure 2 Dynamic light scattering results were depicted for L-arginine titration of 50 mg / mL buffer-exchanged ravoizumab (ALXN1210).

[0063] Figure 3 Dynamic light scattering results were depicted for salt titration of 50 mg / mL phosphate-buffered saline-exchanged ravoizumab (ALXN1210).

[0064] Figure 4 Differential scanning fluorescence results were depicted for 50 mg / mL buffer-exchanged ravoizumab (ALXN1210).

[0065] Figure 5 Dynamic light scattering results were depicted for 10 mg / mL and 114 mg / mL (without L-arginine) and 114 mg / mL (with L-arginine).

[0066] Figure 6 Stability data for ravezozine (ALXN1210) are shown (T=0 to T=2 weeks at 2-8°C).

[0067] Figure 7 Stability data for ravezozumab (ALXN1210) were shown (at 2-8°C from T=3 weeks to T=2 months).

[0068] Figure 8 Stability data for ravezozumab (ALXN1210) are shown (at 23-27°C for T=0 to T=3 weeks).

[0069] Figure 9Stability data for ravezozumab (ALXN1210) were shown (at 23-27°C for T=1 to T=2 months).

[0070] Figure 10 Stability data for ravezozumab (ALXN1210) are shown (at 37°C from T=1 week to T=3 weeks).

[0071] Figure 11 Stability data for ravezozumab (ALXN1210) were shown (at 37°C for T = 1 month to T = 2 months).

[0072] Figure 12 Stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=2 months at 2-8°C).

[0073] Figure 13 Stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=2 months at 23-27°C).

[0074] Figure 14 Stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=2 months at 37°C).

[0075] Figure 15 Dynamic light scattering data (T=0) for the stability of ravocil (ALXN1210) and histidine samples are shown.

[0076] Figure 16 Dynamic light scattering data (T=0) of the stability of ravocil (ALXN1210) histidine AS samples are shown.

[0077] Figure 17 Dynamic light scattering data (T=0) of the stability of ravocil (ALXN1210) phosphate samples are shown.

[0078] Figure 18 Dynamic light scattering data of the stability of ravocil (ALXN1210) phosphate samples are shown (T = 2 months at 2-8°C).

[0079] Figure 19 The freeze-thaw stability data of ravenizumab (ALXN1210) are shown (cycle 2 from T=0 to T=1 month at -20°C).

[0080] Figure 20The data show the stability of ravocil (ALXN1210) during freeze-thaw cycles 3 to 5 at -20°C for 1 month.

[0081] Figure 21 Stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (freeze-thawed at -20°C for T=1 month).

[0082] Figure 22 Prototype stability data for ravezozumab (ALXN1210) are shown (T=0 to T=2 months at 2-8°C).

[0083] Figure 23 Prototype stability data for ravezozumab (ALXN1210) are shown (T=3 months to T=6 months at 2-8°C).

[0084] Figure 24 Prototype stability data for ravezozumab (ALXN1210) are shown (T=9 months to T=12 months at 2-8°C).

[0085] Figure 25 The prototype stability data of ravezozine (ALXN1210) are shown (T=1 month to T=2 months at 23-27°C).

[0086] Figure 26 The prototype stability data for ravezozumab (ALXN1210) are shown (at 23-27°C for T=3 months to T=6 months).

[0087] Figure 27 Prototype stability data for ravezozumab (ALXN1210) are shown (T=9 months to T=12 months at 23-27°C).

[0088] Figure 28 Prototype stability data for ravezozine (ALXN1210) are shown (T=2 weeks to T=2 months at 37°C).

[0089] Figure 29 Prototype stability data for ravezozine (ALXN1210) are shown (T=1 month to T=3 months at -20°C).

[0090] Figure 30 Prototype stability data for ravezozumab (ALXN1210) are shown (T=6 months to T=12 months at -20°C).

[0091] Figure 31The results of the prototype stability study of raveizumab (ALXN1210) are shown (at -80°C for T=3 months to T=6 months).

[0092] Figure 32 The results of the prototype stability study of ravezozumab (ALXN1210) are shown (T=12 months at -80°C).

[0093] Figure 33 Prototype stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravoizumab (ALXN1210) (T=0 to T=12 months at 2-8°C).

[0094] Figure 34 Prototype stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=12 months at 23-27°C).

[0095] Figure 35 Prototype stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=12 months at 37°C).

[0096] Figure 36 Prototype stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravocil (ALXN1210) (T=0 to T=12 months at -20°C).

[0097] Figure 37 Prototype stability size exclusion chromatography (SEC) data are shown, i.e., monomer % of ravoizumab (ALXN1210) (T=0 to T=12 months at -80°C).

[0098] Figure 38 Prototype stability dynamic light scattering data (T=0) of 75 mg / mL ravoizumab ALXN1210 phosphate sample are shown.

[0099] Figure 39 Prototype stability dynamic light scattering data of 75 mg / mL ramucirumab ALXN1210 phosphate sample are shown (T = 1 month at 2-8°C).

[0100] Figure 40 The prototype stability dynamic light scattering data of 100 mg / mL ravocil (ALXN1210 phosphate sample) are shown (T = 1 month at 2-8°C).

[0101] Figure 41The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles of -20°C for 1 month 1-3.

[0102] Figure 42 The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles of -20°C for 1 month 4-5.

[0103] Figure 43 The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles 1-3 at -80°C for T=3 months.

[0104] Figure 44 The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles of -80°C for 3 months 4-5.

[0105] Figure 45 The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles 1-5 at -20°C for T=1 month.

[0106] Figure 46 The prototype stability size exclusion chromatography (SEC) data are shown, namely monomer % of ravocil (ALXN1210) under freeze-thaw cycles 1-5 at -80°C for T=3 months.

[0107] Figure 47 The overall design of the Phase 1 study is described, which is designed to evaluate the safety, tolerability, pharmacokinetics, PD, and immunogenicity of a single subcutaneous dose of 400 mg levofloxacin (ALXN1210) compared with a single intravenous dose of 400 mg levofloxacin (ALXN1210) or a subcutaneous placebo in 42 healthy subjects.

[0108] Figure 48 An overview of the treatment of all subjects is provided.

[0109] Figure 49 This is a graph that uses linear scaling to depict the relationship between individual ALXN1210 serum concentrations and nominal time.

[0110] Figure 50 It is a graph that uses a log-linear scaling method to depict the relationship between individual ALXN1210 serum concentrations and nominal time.

[0111] Figure 51This is a graph depicting the mean (±SD) percentage change over time of free C5 serum concentrations relative to baseline in subjects receiving placebo SC, ALXN1210 SC, and ALXN1210 IV.

[0112] Figure 52 This is a graph depicting the mean (±SD) percentage change in total C5 serum concentration over time relative to baseline in subjects who received placebo SC, ALXN1210 SC, and ALXN1210 IV.

[0113] Figure 53 This is a graph depicting the change in the mean (±SD) percentage of chicken red blood cell (cRBC) hemolysis over time relative to baseline in subjects who received placebo SC, ALXN1210 SC, and ALXN1210 IV. Detailed Implementation

[0114] This disclosure specifically reports stable aqueous solutions containing high concentrations of anti-C5 antibodies (e.g., renjuzumab). These solutions can be used in a variety of therapeutic applications, such as methods for treating or preventing complement-related conditions. While not intended to be limiting, exemplary solutions, formulations, therapeutic kits, and methods for preparing and using any of the aforementioned substances are described in detail below, and illustrated by examples in working embodiments.

[0115] I. definition

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although similar or equivalent methods and compositions to those described herein may be used in the practice or testing of the invention, preferred methods and compositions are described herein.

[0117] Unless the context clearly indicates otherwise, the singular forms “a,” “a,” and “the” include plural indicators.

[0118] The term “about,” especially in relation to a given quantity or number, is intended to cover deviations within plus or minus ten percent (±10%) (e.g., ±5%).

[0119] The term "pharmaceutical formulation" refers to a formulation in which the bioactivity of the active ingredient is clearly effective and which does not contain any other components that would be obviously toxic to the subject to which the formulation is to be administered.

[0120] As used herein, an "aqueous" pharmaceutical composition is a composition suitable for pharmaceutical use, wherein the aqueous carrier is water. A pharmaceutically suitable composition may be sterile, homogeneous, and / or isotonic. Aqueous pharmaceutical compositions may be prepared directly in an aqueous form and / or may be reconstituted from lyophilized material.

[0121] An "isotonic" formulation is one that has an osmotic pressure substantially the same as that of human blood. Isotonic formulations typically have an osmotic pressure of approximately 275 to 350 mOsm / kg. The term "hypotonic" describes a formulation with an osmotic pressure lower than that of human blood. Correspondingly, the term "hypertonic" is used to describe a formulation with an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure meter or a cryo-osmometer. A "straining agent" is a compound that makes a formulation isotonic.

[0122] As used herein, the “molar osmolality” of a solution is the number of osmotic moles of solute per kilogram of solvent. Molar osmolality is a measure of the number of particles present in a solution and is independent of particle size or weight. It can only be measured using solution properties that depend solely on particle concentration. These properties are vapor pressure depression, freezing point depression, boiling point elevation, and osmotic pressure, collectively referred to as colligative properties.

[0123] "Sterile" preparations are sterile or contain little or no living microorganisms or their spores.

[0124] As used herein, a “stable” formulation is one in which the antibody substantially retains its physical and / or chemical stability and / or biological activity after storage. Various analytical techniques for measuring protein stability are available in the art and have been reviewed in Peptide and Protein Drug Delivery, 247-301, edited by Vincent Lee, Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability of anti-C5 antibody formulations can be measured at selected temperatures after a selected time period. For example, increased aggregate formation after storage is an indicator of instability in aqueous anti-C5 antibody formulations. In addition to aggregate formation, retention of original transparency, color, and odor throughout the shelf life is also an indicator used to monitor the stability of aqueous anti-C5 antibody solutions described herein.

[0125] If an antibody shows virtually no signs of aggregation, precipitation, and / or denaturation when visually inspected for color and / or transparency, or as measured by ultraviolet light scattering or size exclusion chromatography, then the antibody “maintains its physical stability” in the pharmaceutical formulation.

[0126] The term "aggregation" refers to the assembly of naturally folded proteins into aggregates containing non-natural structures. Aggregation can occur even under physiological, non-denaturing conditions and is often irreversible, resulting in inactive, sometimes immunogenic and toxic, non-natural aggregates.

[0127] As used herein, the phrase “low to undetectable aggregation levels” refers to aggregates in a sample that, as measured by gel permeation high-performance liquid chromatography (GP-HPLC), high-performance size exclusion chromatography (HPSEC), or static light scattering (SLS) techniques, do not exceed about 5%, about 4%, about 3%, about 2%, about 1%, and about 0.5% by weight of protein.

[0128] As described below, an antibody is considered to retain its biological activity if its chemical stability at a given time is sufficient. Chemical stability can be assessed by detecting and quantifying chemical alterations of the antibody. These alterations can involve size changes (e.g., shearing), deamidation, racemization, hydrolysis, oxidation, β-elimination, and disulfide exchange, and can be evaluated using known techniques such as size exclusion chromatography, SDS-PAGE, matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS), and / or ion exchange chromatography.

[0129] If an antibody in a pharmaceutical formulation is biologically active for its intended purpose, then the antibody “retains its biological activity” in the pharmaceutical formulation. For example, if the biological activity of an antibody in a pharmaceutical formulation is within about 30%, about 20%, or about 10% (within the measurement error range) of the biological activity exhibited during the preparation of the pharmaceutical formulation (e.g., as measured in an antigen binding assay). In this document, “biological activity” of a monoclonal antibody refers to the ability of the antibody to bind to an antigen. It may also include antibodies that bind to an antigen and produce a measurable biological response, which can be measured in vitro or in vivo.

[0130] The shelf life of a pharmaceutical product (e.g., an aqueous solution containing anti-C5 antibodies) is the length of time the product is stored before it decomposes. For example, shelf life can be defined as the time it takes for 0.1%, 0.5%, 1%, 5%, or 10% of the product to decompose.

[0131] As used herein, the term "antibody" describes a polypeptide containing at least one antibody-derived antigen-binding site (e.g., a VH / VL region or Fv or CDR). Antibodies include antibodies in known forms. For example, an antibody can be a human antibody, a humanized antibody, a bispecific antibody, or a chimeric antibody. Antibodies can also be Fab, Fab'2, ScFv, SMIP, Affibody, nanobody, or domain antibody. Antibodies can also be any of the following isotypes: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE. Antibodies can be naturally occurring antibodies or antibodies that have been modified by protein engineering techniques (e.g., through mutation, deletion, substitution, or conjugation to non-antibody moieties). For example, an antibody may include one or more variant amino acids that alter antibody properties (e.g., functional properties) compared to naturally occurring antibodies. For example, many such modifications are known in the art that affect, for example, half-life, effector function, and / or the immune response to antibodies in a patient. The term antibody also includes artificially or engineered polypeptide constructs containing at least one antigen-binding site of antibody origin.

[0132] As used herein, the terms "specific binding," "selective binding," "selectively binding," and "specific binding" refer to an antibody binding to an epitope on a predetermined antigen without binding to other antigens. Typically, antibodies (i) bind to epitopes on a predetermined antigen using, for example, surface plasmon resonance (SPR) technology. When a predetermined antigen (e.g., C5) is used as the analyte and the antibody as the ligand in a 2000 surface plasmon resonance instrument, or when the assay is performed by Scatchard analysis of antibody-antigen-positive cells, the equilibrium dissociation constant (K0) of the binding is determined. D Approximately less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower, and (ii) the affinity for the predetermined antigen is at least twice that for nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. Therefore, unless otherwise stated, an antibody that "specifically binds to human C5" refers to an antibody with an affinity of 10... -7 M or smaller, such as approximately less than about 10 -8 M, 10 -9 M or 10 -10 M or even lower K D Antibodies that bind to human C5, which are soluble or cellular.

[0133] As used herein, the term “surface plasmon resonance” refers to an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration in a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, U. et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U. et al. (1991) Biotechniques 11: 620-627; Johnson, B. et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnson, B. et al. (1991) Anal. Biochem. 198: 268-277.

[0134] As used in this article, the term "K" off "This refers to the dissociation rate constant of the antibody from the antibody / antigen complex."

[0135] As used in this article, the term "K" d "This refers to the dissociation constant of a specific antibody-antigen interaction."

[0136] As used herein, the terms “subject” or “patient” are used interchangeably and refer to mammals such as humans, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, monkeys, cattle, horses, pigs, etc. In one implementation, a patient is a human patient (e.g., a human patient with complement-related symptoms).

[0137] As used herein, the term “treatment” refers to the therapeutic measures described herein. A “treatment” method involves administering to a subject a combination of the treatments disclosed herein in order to cure or delay a disease or condition or a recurrence of a disease or condition, reduce its severity, or improve one or more of its symptoms, or to prolong the subject’s survival beyond what would have been expected without such treatment.

[0138] As used herein, "effective treatment" means treatment that produces a beneficial effect, such as improving at least one symptom of a disease or condition. A beneficial effect can manifest as improvement relative to a baseline, i.e., improvement relative to measurements or observations made prior to initiating treatment according to the described method. Effective treatment can also refer to the reduction of at least one symptom of a disease or condition.

[0139] The term "effective amount" refers to the amount of a pharmaceutical agent that provides a desired biological, therapeutic, and / or preventive outcome. This outcome may be a reduction, improvement, alleviation, ablation, delay, and / or alleviation of one or more signs, symptoms, or causes of a disease or condition, or any other desired alteration of a biological system. In one instance, an "effective amount" is the amount of a stable aqueous solution that alleviates at least one symptom of a disease or condition. An effective amount may be administered in a single or multiple-dose manner.

[0140] As used in this article, the terms “induction” and “induction phase” are used interchangeably and refer to the first phase of treatment.

[0141] As used herein, the terms “maintenance” and “maintenance phase” are used interchangeably and refer to the second phase of treatment. In some implementations, treatment continues as long as clinical benefit is observed or until unmanageable toxicity or disease progression occurs.

[0142] II. anti-C5 antibody

[0143] The anti-C5 antibodies described herein bind to complement component C5 (e.g., human C5) and inhibit C5 cleavage into fragments C5a and C5b. As mentioned above, these antibodies also possess, for example, improved pharmacokinetic properties compared to other anti-C5 antibodies used for therapeutic purposes (e.g., eculizumab).

[0144] Suitable anti-C5 antibodies (or VH / VL domains derived therefrom) for use in this invention can be produced using methods well known in the art. Alternatively, anti-C5 antibodies recognized in the art can be used. Antibodies that competitively bind to C5 with any of these recognized antibodies can also be used.

[0145] An exemplary anti-C5 antibody is rivuizumab, which comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 14 and 11, respectively, or antigen-binding fragments and variants thereof. Ramuizumab (also known as BNJ441 and ALXN1210) is described in PCT / US2015 / 019225 and US Patent No. 9,079,949, the teachings of which are hereby incorporated by reference. Throughout this document, the terms rivuizumab, BNJ441, and ALXN1210 are used interchangeably. Ramuizumab selectively binds to human complement protein C5, inhibiting its cleavage into C5a and C5b during complement activation. This inhibition prevents the release of the pro-inflammatory mediator C5a and the formation of the cytolytic pore-forming membrane attack complex (MAC) C5b-9, while preserving the proximal or early components of complement activation (e.g., C3 and C3b) necessary for microbial opsonization and immune complex clearance.

[0146] In other embodiments, the antibody comprises heavy and light chain CDR or variable regions of revoizumab. Thus, in one embodiment, the antibody comprises CDR1, CDR2, and CDR3 domains of the VH region of revoizumab having the sequence shown in SEQ ID NO: 12, and CDR1, CDR2, and CDR3 domains of the VL region of revoizumab having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 4, 5, and 6, respectively. In yet another embodiment, the antibody comprises VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0147] Another exemplary anti-C5 antibody is antibody BNJ421, which comprises a heavy chain and a light chain having the sequences shown in SEQ ID NO: 20 and 11, respectively, or antigen-binding fragments and variants thereof. BNJ421 (also known as ALXN1211) is described in PCT / US2015 / 019225 and U.S. Patent No. 9,079,949, the teachings of which are hereby incorporated by reference.

[0148] In other embodiments, the antibody comprises heavy and light chain CDR or variable regions of BNJ421. Thus, in one embodiment, the antibody comprises CDR1, CDR2, and CDR3 domains of the VH region of BNJ421 having the sequence shown in SEQ ID NO: 12, and CDR1, CDR2, and CDR3 domains of the VL region of BNJ421 having the sequence shown in SEQ ID NO: 8. In another embodiment, the antibody comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 19, 18, and 3, respectively, and light chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NO: 4, 5, and 6, respectively. In yet another embodiment, the antibody comprises VH and VL regions having the amino acid sequences shown in SEQ ID NO: 12 and SEQ ID NO: 8, respectively.

[0149] The exact boundaries of CDRs have been defined differently according to various methods. In some implementations, the location of a CDR or framework region within a variable domain of the light or heavy chain can be as defined by Kabat et al. [(1991) "Sequences of Proteins of Immunological Interest." NIH Publication No. 91-3242, Department of Health and Human Services, Bethesda, MD]. In such cases, the CDR can be referred to as a "Kabat CDR" (e.g., "Kabat LCDR2" or "Kabat HCDR1"). In some implementations, the location of a CDR within a variable region of the light or heavy chain can be as defined by Chothia et al. (1989) in Nature. 342 As defined in 877-883. Therefore, these regions can be referred to as “Chothia CDR” (e.g., “Chothia LCDR2” or “Chothia HCDR3”). In some embodiments, the CDR locations of the light and heavy chain variable regions can be as defined in the Kabat-Chothia combination definition. In such embodiments, these regions can be referred to as “combined Kabat-Chothia CDR”. Thomas et al. [(1996) Mol Immunol 33(17 / 18) [1389-1401] Examples illustrate the identification of CDR boundaries according to the definitions of Kabat and Chothia.

[0150] In some embodiments, the anti-C5 antibody described herein comprises a heavy chain CDR1, which contains or consists of the following amino acid sequence: G H IFSNYWIQ (SEQ ID NO: 19). In some embodiments, the anti-C5 antibody described herein comprises a heavy chain CDR2, which contains or consists of the following amino acid sequence: EILPGSG H TEYTENFKD (SEQ ID NO: 18). In some embodiments, the anti-C5 antibody described herein comprises a heavy chain variable region containing the following amino acid sequence:

[0151] QVQLVQSGAEVKKPGASVKVSCKASG H IFSNYWIQWVRQAPGQGLEWMGEILPGSG HTEYTENFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYFFGSSPNWYFDVWGQGTLVTVSS (SEQ ID NO: 12).

[0152] In some embodiments, the anti-C5 antibody described herein comprises a light chain variable region containing the following amino acid sequence:

[0153] DIQMTQSPSSSLSASVGDRVTITTCGASENIYGALNWYQQKPGKAPKLLIYGATNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNVLNTPLTFGQGTKVEIK (SEQ ID NO: 8).

[0154] In some embodiments, the anti-C5 antibody described herein may comprise a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn) with a greater affinity than the native human Fc constant region from which the variant human Fc constant region is derived. For example, the Fc constant region may contain one or more (e.g., two, three, four, five, six, seven, or eight or more) amino acid substitutions relative to the native human Fc constant region from which the variant human Fc constant region is derived. Such substitutions may increase the binding affinity of the IgG antibody containing the variant Fc constant region to FcRn at pH 6.0 while maintaining the pH dependence of the interaction. Methods for testing whether one or more substitutions in the antibody Fc constant region increase the affinity of the Fc constant region to FcRn at pH 6.0 (while maintaining the pH dependence of the interaction) are known in the art and are exemplified in working examples. See, for example, PCT / US2015 / 019225 and U.S. Patent No. 9,079949, the disclosures of which are incorporated herein by reference in their entirety.

[0155] Substitution of the antibody Fc constant region to enhance the binding affinity of FcRn is known in the art, including, for example, (1) Dall'Acqua et al. (2006) J Biol Chem 281 : 23514-23524 describes the M252Y / S254T / T256E triple substitution; (2) in Hinton et al. (2004) J Biol Chem 279 :6213-6216 and Hinton et al. (2006) J Immunol 176 The substitution of M428L or T250Q / M428L as described in 346-356; (3) in Petkova et al. (2006) Iht Immunol 18(12)The substitutions described in 1759-69 are N434A or T307 / E380A / N434A. Other substitution pairs: P257I / Q311I, P257I / N434H and D376V / N434H are, for example, in Datta-Mannan et al. (2007) J Biol Chem 282(3) The description is found in 1709-1717, and its public content is incorporated herein by reference in its entirety.

[0156] In some embodiments, the variant constant region has a valine substitution at EU amino acid residue 255. In some embodiments, the variant constant region has an asparagine substitution at EU amino acid residue 309. In some embodiments, the variant constant region has an isoleucine substitution at EU amino acid residue 312. In some embodiments, the variant constant region has a substitution at EU amino acid residue 386.

[0157] In some embodiments, the variant Fc constant region contains no more than 30 amino acid substitutions, insertions, or deletions (e.g., no more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2) relative to its derived natural constant region. In some embodiments, the variant Fc constant region contains one or more amino acid substitutions selected from the group consisting of M252Y, S254T, T256E, N434S, M428L, V259I, T250I, and V308F. In some embodiments, the variant human Fc constant region contains methionine at position 428 and asparagine at position 434, each according to EU designations. In some implementations, the variant Fc constant region contains 428L / 434S double substitution, as described, for example, in U.S. Patent No. 8,088,376.

[0158] In some implementations, the precise location of these mutations can be offset from the location of the natural human Fc constant region due to antibody engineering. For example, when used in IgG2 / 4 chimeric Fc, the 428L / 434S double substitution can correspond to 429L and 435S, as in the M429L and N435S variants found in revozumab (BNJ441) and described in U.S. Patent No. 9,079,949, the disclosure of which is incorporated herein by reference in its entirety.

[0159] In some embodiments, the variant constant region contains substitutions relative to the natural human Fc constant region at amino acid positions 237, 238, 239, 248, 250, 252, 254, 255, 256, 257, 258, 265, 270, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317, 325, 332, 334, 360, 376, 380, 382, ​​384, 385, 386, 387, 389, 424, 428, 433, 434, or 436 (EU number). In some embodiments, the substitution is selected from the group consisting of: methionine replacing glycine at position 237; alanine replacing proline at position 238; lysine replacing serine at position 239; isoleucine replacing lysine at position 248; alanine, phenylalanine, isoleucine, methionine, glutamine, serine, valine, tryptophan, or tyrosine replacing threonine at position 250; phenylalanine, tryptophan, or tyrosine replacing methionine at position 252; threonine replacing serine at position 254; and glutamic acid replacing arginine at position 255. At position 256, aspartic acid, glutamic acid, or glutamine replaces threonine; at position 257, alanine, glycine, isoleucine, leucine, methionine, asparagine, serine, threonine, or valine replaces proline; at position 258, histidine replaces glutamic acid; at position 265, alanine replaces aspartic acid; at position 270, phenylalanine replaces aspartic acid; at position 286, alanine or glutamic acid replaces asparagine; at position 289, histidine replaces threonine; at position 297, alanine replaces asparagine; at position 298, glycine replaces serine. At position 303, alanine replaces valine; at position 305, alanine replaces valine; at position 307, alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine replace threonine; at position 308, alanine, phenylalanine, isoleucine, leucine, methionine, proline, glutamine, or threonine replace valine; at position 309, alanine, aspartic acid, glutamic acid, proline, or arginine replaces valine. Substitute leucine or valine; at position 311, glutamine is replaced by alanine, histidine, or isoleucine; at position 312, aspartic acid is replaced by alanine or histidine; at position 314, leucine is replaced by lysine or arginine; at position 315, asparagine is replaced by alanine or histidine; at position 317, lysine is replaced by alanine; at position 325, asparagine is replaced by glycine; at position 332, isoleucine is replaced by valine; at position 334, lysine is replaced by leucine; at position 360, lysine is replaced by histidine; at position 376, aspartic acid is replaced by alanine.At position 380, alanine replaces glutamic acid; at position 382, ​​alanine replaces glutamic acid; at position 384, alanine replaces asparagine or serine; at position 385, aspartic acid or histidine replaces glycine; at position 386, proline replaces glutamine; at position 387, glutamic acid replaces proline; at position 389, alanine or serine replaces asparagine; at position 424, alanine replaces serine; at position 428, alanine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, or tyrosine replace methionine; at position 433, lysine replaces histidine; at position 434, alanine, phenylalanine, histidine, serine, tryptophan, or tyrosine replace asparagine; and at position 436, histidine replaces tyrosine or phenylalanine, all according to EU designations.

[0160] In some embodiments, a suitable anti-C5 antibody used in the methods described herein comprises a heavy chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 14 and / or a light chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 11. Alternatively, in some embodiments, the anti-C5 antibody used in the methods described herein comprises a heavy chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 20 and / or a light chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 11.

[0161] In one implementation, the antibody binds to C5 at 7.4 and 25 °C (and additionally under physiological conditions), its affinity dissociation constant (K5) is determined. D The concentration is at least 0.1 (e.g., at least 0.15, 0.175, 0.2, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, or 0.975) nM. In some embodiments, the K of the anti-C5 antibody or its antigen-binding fragment is... D Not greater than 1 (e.g., not greater than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 or 0.2) nM.

[0162] In other embodiments, [(the antibody is effective against the K+ of C5 at pH 6.0 and 25°C)] D ) / (Antibody against C5 K at pH 7.4 and 25°C DGreater than 21 (e.g., greater than 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 2) 40, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, or 8000).

[0163] Methods for determining whether an antibody binds to a protein antigen and / or measuring the antibody's affinity for the protein antigen are known in the art. For example, a variety of techniques can be used to detect and / or quantify antibody binding to a protein antigen, such as, but not limited to, Western blot, dot blot, surface plasmon resonance (SPR) methods (e.g., the BIAcore system; Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ), or enzyme-linked immunosorbent assay (ELISA). See, for example, Benny KCLo (2004) "Antibody Engineering: Methods and Protocols," Humana Press (ISBN: 1588290921); Johne et al. (1993) J ImmunolMeth 160 : 191-198; Jonsson et al. (1993) Ann Biol Clin 51 : 19-26; and Jonsson et al. (1991) Biotechniques 11 References: 620-627. Additionally, methods for measuring affinity (e.g., dissociation constant and association constant) are presented in the working embodiments.

[0164] As used in this article, the term "k" a "k" refers to the rate constant of antibody-antigen association. The term "k" is... d "K" refers to the rate constant of antibody dissociation from the antibody / antigen complex. The term "K" is also used in this context. D "" refers to the equilibrium dissociation constant of antibody-antigen interactions. The equilibrium dissociation constant is the ratio of the kinetic rate constant K to the equilibrium dissociation constant. D =k a / k dThis is derived. Such measurements are preferably taken at 25°C or 37°C (see working examples). For example, antibodies can be immobilized using an anti-Fc capture method on a BIAcore 3000 instrument, and the kinetics of antibodies binding to human C5 can be determined by surface plasmon resonance (SPR) at pH 8.0, 7.4, 7.0, 6.5, and 6.0.

[0165] In one embodiment, an anti-C5 antibody or its antigen-binding fragment blocks the production or activity of the C5a and / or C5b active fragments of the C5 protein (e.g., human C5 protein). Through this blocking action, the antibody inhibits, for example, the pro-inflammatory effects of C5a and the production of the cell surface C5b-9 membrane attack complex (MAC).

[0166] Methods for determining whether a particular antibody described herein inhibits C5 cleavage are known in the art. Inhibition of human complement component C5 reduces the cytolytic capacity of complement in the body fluids of a subject. Such a reduction in the cytolytic capacity of complement present in body fluids can be measured by methods well known in the art, such as by routine hemolysis assays, such as those described in Kabat and Mayer (ed.), “Experimental Immunochemistry, 2nd ed.” 135-240, Springfield, IL, C. Thomas (1961), pp. 135-139, or routine variations of such assays, such as those described in, for example, Hillmen et al. (2004) N Engl J Med. 350(6) The method for hemolysis of chicken erythrocytes described in :552. Methods for determining whether a candidate compound inhibits the cleavage of human C5 cells into C5a and C5b forms are known in the art and are described in Evans et al. (1995) Mol Immunol 32(16) As described in 1183-95. For example, the concentrations and / or physiological activities of C5a and C5b in body fluids can be measured using methods well known in the art. For C5b, hemolysis assays or assays for soluble C5b-9 as discussed herein can be used. Other assays known in the art can also be used. Using these or other suitable types of assays, candidate agents capable of inhibiting human complement component C5 can be screened.

[0167] Immunological techniques (such as, but not limited to, ELISA) can be used to measure the protein concentration of C5 and / or its cleavage products to determine the ability of anti-C5 antibodies or their antigen-binding fragments to inhibit the conversion of C5 into its biologically active product. In some embodiments, the production of C5a is measured. In some embodiments, a novel epitope-specific antibody against C5b-9 is used to detect the formation of terminal complement.

[0168] A hemolysis assay can be used to determine the inhibitory activity of anti-C5 antibodies or their antigen-binding fragments against complement activation. To determine the effect of anti-C5 antibodies or their antigen-binding fragments on hemolysis mediated by the classical complement pathway in in vitro serum test solutions, for example, hemolysin-coated sheep erythrocytes or chicken erythrocytes sensitized with anti-chicken erythrocyte antibodies are used as target cells. The percentage of lysis is normalized by considering 100% lysis as equal to lysis occurring in the absence of inhibitors. In some embodiments, the classical complement pathway is activated by human IgM antibodies, for example, as in... Classical pathway complement kit ( The method described in COMPL CP310 (Euro-Diagnostica, Sweden) involves incubating test serum with an anti-C5 antibody or its antigen-binding fragment in the presence of a human IgM antibody. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorescent substrate, and measuring the absorbance at an appropriate wavelength. As a control, the test serum is incubated in the absence of an anti-C5 antibody or its antigen-binding fragment. In some embodiments, the test serum is a C5-deficient serum reconstituted with a C5 peptide.

[0169] To determine the effect of anti-C5 antibodies or their antigen-binding fragments on alternative pathway-mediated hemolysis, unsensitized rabbit or guinea pig erythrocytes can be used as target cells. In some embodiments, the serum test solution is C5-deficient serum reconstituted with a C5 peptide. The percentage of lysis is normalized by considering 100% lysis as equal to lysis occurring in the absence of inhibitors. In some embodiments, the alternative complement pathway is activated by lipopolysaccharide molecules, for example, as in... Alternative Pathway Complement Kit ( The method used in COMPL AP330 (Euro-Diagnostica, Sweden) involves incubating test serum with an anti-C5 antibody or its antigen-binding fragment in the presence of lipopolysaccharide. The amount of C5b-9 produced is measured by contacting the mixture with an enzyme-conjugated anti-C5b-9 antibody and a fluorescent substrate, and measuring fluorescence at an appropriate wavelength. As a control, the test serum is incubated in the absence of an anti-C5 antibody or its antigen-binding fragment.

[0170] In some implementations, the CH50eq assay is used to quantify C5 activity or its inhibition. The CH50eq assay is a method for measuring total classical complement activity in serum. This assay is a lysis assay that uses antibody-sensitized erythrocytes as activators of the classical complement pathway and determines the amount (CH50) required to produce 50% lysis using various test serum dilutions. The percentage of hemolysis can be determined, for example, using a spectrophotometer. The CH50eq assay provides a measure of splicing of terminal complement complex (TCC) formation, as TCC itself is the direct cause of the hemolysis being measured.

[0171] This assay is well known and commonly practiced by those skilled in the art. In short, to activate the classical complement pathway, an undiluted serum sample (e.g., a reconstituted human serum sample) is added to micro-assay wells containing antibody-sensitized red blood cells, thereby generating TCC. Next, the activated serum is diluted in the micro-assay wells, which are coated with a capture reagent (e.g., an antibody that binds to one or more components of the TCC). The TCC present in the activated sample binds to the monoclonal antibody coated on the surface of the micro-assay well. The wells are washed, and a detection reagent is added to each well, which is detectably labeled and identifies the bound TCC. The detectable label can be, for example, a fluorescent label or an enzyme label. Assay results are expressed as CH50 unit equivalents per milliliter (CH50 U Eq / mL).

[0172] Inhibition, for example because it is related to terminal complement activity, includes, for example, a reduction of at least 5% (e.g., at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%) in the activity of the terminal complement compared to that of a control antibody (or its antigen-binding fragment) under similar conditions and at equimolar concentrations in a hemolysis assay or CH50eq assay. As used herein, substantial inhibition means an inhibition of at least 40% (e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher) of a specified activity (e.g., terminal complement activity). In some embodiments, the anti-C5 antibody described herein contains one or more amino acid substitutions relative to the CDR (i.e., SEQ ID NO: 1-6) of eculizumab, and retains at least 30% (e.g., at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) of the complement inhibitory activity of eculizumab in hemolysis assays or CH50eq assays.

[0173] The anti-C5 antibody described herein has a serum half-life in humans of at least 20 days (e.g., at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 days). In another embodiment, the anti-C5 antibody described herein has a serum half-life in humans of at least 40 days. In another embodiment, the anti-C5 antibody described herein has a serum half-life in humans of approximately 43 days. In yet another embodiment, the anti-C5 antibody described herein has a serum half-life in humans between 39 and 48 days. Methods for measuring the serum half-life of the antibody are known in the art. In some embodiments, for example, as measured in a mouse model system (e.g., a C5-deficient / NOD / scid mouse or an hFcRn transgenic mouse model system) as described in the working embodiments, the serum half-life of the anti-C5 antibody or its antigen-binding fragment described herein is at least 20% longer than that of eculizumab (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%).

[0174] In one embodiment, the antibody competes with the antibody described herein for binding to and / or binding to the same epitope on C5. The term “binding to the same epitope” with respect to two or more antibodies means, as determined by a given method, that the antibody binds to the same segment of amino acid residues. Techniques for determining whether an antibody binds to the “same epitope on C5” with the antibody described herein include, for example, epitope mapping methods, such as X-ray analysis of antigen:antibody complex crystals (providing atomic resolution of the epitope) and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to peptide antigen fragments or mutant variants of the antigen, where loss of binding due to modification of amino acid residues within the antigen sequence is generally considered an indicator of epitope composition. Alternatively, computational combinatorial methods for epitope mapping may also be used. These methods rely on the ability of the target antibody to affinity-isolate specific short peptides from a combinatorial phage display peptide library. Antibodies having the same VH and VL or the same CDR1, 2, and 3 sequences are expected to bind to the same epitope.

[0175] An antibody that “competes with another antibody to bind to a target” is one that inhibits (partially or completely) the binding of another antibody to a target. Known competition assays can be used to determine whether two antibodies compete with each other to bind to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to the target. In some embodiments, one antibody competes with another antibody and inhibits the binding of the other antibody to the target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The level of inhibition or competition can vary depending on which antibody is a “blocking antibody” (i.e., a cold antibody that is first incubated with the target). Competing antibodies bind to the same epitope, overlapping epitope, or adjacent epitope (e.g., demonstrated by steric hindrance).

[0176] A variety of techniques well-known in the art can be used to generate the anti-C5 antibody or its antigen-binding fragment described herein for use in the methods described herein. Monoclonal antibodies can be obtained using a variety of techniques familiar to those skilled in the art. Briefly, spleen cells from animals immunized with the desired antigen are typically immortalized by fusion with myeloma cells (see, Kohler and Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr virus, oncogenes, or retroviruses, or other methods well-known in the art. Colonies produced by single immortalized cells are screened to produce antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by such cells can be increased by a variety of techniques, including intraperitoneal injection into the vertebrate host. Alternatively, DNA libraries can be screened from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989), thereby isolating DNA sequences encoding monoclonal antibodies or their binding fragments.

[0177] III. Highly concentrated anti-C5 antibody solution

[0178] This document provides stable aqueous solutions containing anti-C5 antibodies (e.g., revoizumab). The aqueous solutions described herein may be sterile, pharmaceutical-grade compositions, for example, intended for administration to subjects to treat or prevent complement-related conditions such as PNH or aHUS. The solutions described herein may be prepared according to standard methods. Pharmaceutical formulations are validated techniques and are further described, for example, in Gennaro (2000), “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams & Wilkins (ISBN: 0683306472); Ansel et al. (1999), “Pharmaceutical Dosage Forms and Drug Delivery Systems,” 7th edition, Lippincott, Williams & Wilkins Publishers (ISBN: 0683305727); and Kibbe (2000), “Handbook of Pharmaceutical Excipients, American Pharmaceutical Association,” 3rd edition (ISBN: 091733096X). The working examples illustrate a suitable method for preparing the high-concentration antibody solution described herein.

[0179] The aqueous solutions described herein contain high concentrations of antibodies (such as revozumab) that bind to human complement component C5. Such solutions are sometimes referred to herein as “high-concentration antibody solutions.” As used herein, a “high concentration” of anti-C5 antibody (e.g., ravoizumab) in aqueous solution is an antibody concentration of at least, equal to, or greater than 40 (e.g., at least, equal to, or greater than 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300) mg / mL. In one embodiment, the concentration of anti-C5 antibody in solution is greater than 100 (e.g., greater than 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, or 195) mg / mL. In another embodiment, the concentration of anti-C5 antibody in solution is greater than 200 (e.g., greater than 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, or 295) mg / mL. In yet another embodiment, the concentration of anti-C5 antibody in solution is greater than 300 mg / mL.In another embodiment, the antibody is present in solution at concentrations, for example, 40 mg / mL to 200 mg / mL, 50 mg / mL to 200 mg / mL, 60 mg / mL to 200 mg / mL, 70 mg / mL to 200 mg / mL, 80 mg / mL to 200 mg / mL, 90 mg / mL to 200 mg / mL, 100 mg / mL to 200 mg / mL, 110 mg / mL to 200 mg / mL, 120 mg / mL to 200 mg / mL, 130 mg / mL to 200 mg / mL, 140 mg / mL to 200 mg / mL, 150 mg / mL. g / mL to 200 mg / mL, 40 mg / mL to 100 mg / mL, 50 mg / mL to 100 mg / mL, 60 mg / mL to 100 mg / mL, 70 mg / mL to 100 mg / mL, 80 mg / mL to 100 mg / mL, 90 mg / mL to 100 mg / mL, 40 mg / mL to 150 mg / mL, 50 mg / mL to 150 mg / mL, 60 mg / mL to 150 mg / mL, 70 mg / mL to 150 mg / mL, 80 mg / mL to 150 mg / mL, 90 mg / mL to 150 mg / mL, 10 0 mg / mL to 150 mg / mL, 110 mg / mL to 150 mg / mL, 120 mg / mL to 150 mg / mL, 40 mg / mL to 50 mg / mL, 40 mg / mL to 250 mg / mL, 50 mg / mL to 250 mg / mL, 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, 100 mg / mL to 250 mg / mL, 110 mg / mL to 250 mg / mL, 120 mg / mL to 250 mg / mL / mL, 130mg / mL to 250mg / mL, 140mg / mL to 250mg / mL, 150mg / mL to 250mg / mL, 160mg / mL to 250mg / mL, 170mg / mL to 250mg / mL, 180mg / mL to 250mg / mL, 190mg / mL to 250mg / mL, 200mg / mL to 250mg / mL, above 200mg / mL (e.g., at least 201mg / mL) to 250mg / mL, or above 200mg / mL (e.g., 201mg / mL or higher) to 300mg / mL.

[0180] As described herein and illustrated by example in the working examples, the specifically reported aqueous solutions provide significant physical and chemical stability, as well as functional stability, for the anti-C5 antibodies formulated therein. For example, the formulations described herein are able to maintain the structural integrity of anti-C5 antibodies (e.g., revozumab) present in high concentrations in solution. In one embodiment, the solution is suitable for storage at 2–8°C (e.g., 4°C). In another embodiment, the solution is formulated for storage at temperatures below 0°C (e.g., -20°C or -80°C). In another embodiment, the solution is formulated for storage at 2–8°C (e.g., 4°C) for up to three years (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1 1 / 2 years, 2 years, 2 1 / 2 years, or 3 years). In yet another embodiment, the solution is suitable for storage at 2–8°C (e.g., 4°C) for at least 1, 2, or 3 years.

[0181] As illustrated in the working examples described herein, the solutions described herein are suitable for maintaining approximately 100 mg / mL of anti-C5 antibody primarily in monomeric form for up to two years at approximately 2°C to 8°C. As used herein, if, for example, as determined using size exclusion chromatography-high performance liquid chromatography (SEC-HPLC, such as gel permeation HPLC), the antibody present in the solution is at least 95% (e.g., at least 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97%). If the monomers are 0.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or more, then the anti-C5 antibody prepared at a high concentration in the specially reported aqueous solution is "mainly monomeric" or "mainly in monomeric form". In one embodiment, the anti-C5 antibody in the solution described herein can remain predominantly monomeric after being stored at approximately 2°C to 8°C (e.g., at, for example, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C) for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen ...

[0182] In one embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revolucratabumab) retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least nine months. In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least one year. In yet another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of monomericity, as determined by SEC-HPLC, during storage at 2°C to 8°C for at least 18 months. In another embodiment, the anti-C5 antibody retains at least 95% (e.g., at least 96%, 97%, 98%, or 99%) of its monomer content during storage at 2°C to 8°C for at least two years, as determined by SEC-HPLC.

[0183] In another embodiment, the concentration in the solution is less than 5% (e.g., less than 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4.0%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3.0%, 2.9%, 2.8%, 2.7%, 2.6%). Antibodies of 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% are oligomeric, aggregated, and / or fragmented. As used herein, antibody fragmentation refers to a molecular weight of the intact antibody that is smaller than a misassembled component or degradation product of the intact antibody. Such fragmented forms include, but are not limited to, free single heavy chain polypeptides, dimer heavy chain polypeptides (e.g., disulfide-linked heavy chain polypeptides), dimer heavy chain polypeptides bound to a light chain polypeptide, single heavy chain polypeptides bound to a light chain polypeptide, or further degradation products or fragments of light or heavy chain polypeptides. In some implementations, after storage at 2°C to 8°C for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-four, or more months), less than 2% (e.g., less than 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of antibody aggregation occurs. In some embodiments, less than 1% (e.g., less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the antibody fragments are stored at 2°C to 8°C for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, thirteen, or thirteen months or longer). Methods for determining the amount of monomeric antibody present in solution and the amount of oligomeric, aggregated, or fragmented forms of anti-C5 antibody are described herein and illustrated by example in working examples.For example, technicians can use size exclusion chromatography-high performance liquid chromatography (SEC-HPLC, e.g., gel permeation HPLC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding technique, intrinsic tryptophan fluorescence, non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and differential scanning calorimetry (DSC) to determine the percentage of intact, fragmented, unfolded intermediates and / or aggregated antibody material present in a given solution.

[0184] In one embodiment of any of the solutions described herein, less than 5% of the anti-C5 antibody (e.g., revozumab) aggregates in the solution, as determined by SEC-HPLC (e.g., gel permeation HPLC). In another embodiment, less than 4% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In another embodiment, less than 3% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In another embodiment, less than 2% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC. In yet another embodiment, less than 1% of the anti-C5 antibody aggregates in the solution, as determined by SEC-HPLC.

[0185] As described herein and illustrated by examples in the working examples, the solutions containing anti-C5 antibodies specifically reported herein retain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%) of their biological / functional activity (e.g., ability to bind to human C5) after being stored at 2°C to 8°C for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine ...

[0186] In another embodiment, the anti-C5 antibody (e.g., revoizumab) present in the solution described herein can retain at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of its hemolytic activity after being stored at 2°C to 8°C for at least one month (e.g., at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nine ... Suitable hemolysis assays for determining whether antibodies in a specially reported solution retain their activity are described herein and are known in the art, such as in vitro hemolysis assays using avian or porcine erythrocytes. Suitable methods for evaluating the ability of antibody formulations to bind to human complement component C5 are known in the art and are described herein.

[0187] In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least six months. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least nine months. In another embodiment, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least one year. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least eighteen months. In another embodiment, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least two years. In another embodiment, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its C5 binding activity compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least three years.

[0188] In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least nine months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least six months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least one year. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at 2°C to 8°C for at least 18 months. In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revoizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least two years.In another embodiment of any of the solutions described herein, the anti-C5 antibody (e.g., revelizumab) retains at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of its ability to inhibit hemolysis compared to a reference anti-C5 antibody corresponding to the anti-C5 antibody prior to storage, during storage at a temperature of 2°C to 8°C for at least three years.

[0189] The aqueous solutions described herein may contain one or more common pharmaceutical agents (e.g., one or more excipients and / or additives, such as buffers, sugars or saccharides, salts, surfactants, solubilizers, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelating agents and / or preservatives).

[0190] In one embodiment, the aqueous solution contains one or more buffers. As used herein, the term "buffer" refers to one or more components that, when added to an aqueous solution, protect the solution from pH changes upon the addition of an acid or base or upon dilution with a solvent. In one embodiment, the solution contains at least one or more buffers. Non-limiting examples of typical buffers that may be included in washing solutions include Tris (tris(hydroxymethyl)methylamine), bis-Tris, bis-Trispropane, histidine, triethanolamine, diethanolamine, formate, acetate, MES (2-(N-morpholine)ethanesulfonic acid), phosphate, HEPES (4-2-hydroxyethyl-1-piperazine ethanesulfonic acid), citrate, MOPS (3-(N-morpholine)propanesulfonic acid), TAPS (3{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine (N,N-bis(2-hydroxyethyl)glycine), Tricine (N-tris(hydroxymethyl)methylglycine), TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsine (dimethylarsine)SSC (sodium citrate saline), and sodium phosphate.

[0191] In another embodiment, the buffer is an amino acid. The amino acid may be, for example, selected from histidine (e.g., L-histidine), serine (e.g., L-serine), and glycine (e.g., L-glycine). In another embodiment, the solution contains two or more buffers. In a particular embodiment, the buffer is sodium phosphate. In one embodiment, the specifically reported solution does not contain any free amino acid as a buffer. In another embodiment, the specifically reported solution contains only one free amino acid as a buffer (e.g., histidine). In another embodiment, the specifically reported solution may contain two or more (e.g., two, three, four, five, six, or seven or more) different amino acids as buffers, such as serine and histidine.

[0192] The concentration of the buffer solution is sufficient to maintain the required pH and can be changed, for example, to maintain the isotonicity of the formulation. Typical concentrations of commonly used buffers in parenteral formulations can be found in the following literature: Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2nd Edition, Chapter 5, p. 194; De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Product. In one embodiment, the concentration of one or more buffers in the formulation is from about 10 mM to 300 mM (inclusive). In another embodiment, the solution contains at least one buffer at a concentration of 10 mM to 200 mM (inclusive). In another embodiment, the aqueous solution described herein contains a buffer at a concentration of at least 10 mM (e.g., at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mM or higher).In another embodiment, the aqueous solution contains a buffer concentration of approximately 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, or 10 mM to 150 mM. mM, 15mM to 150mM, 20mM to 150mM, 25mM to 150mM, 30mM to 150mM, 40mM to 150mM, 50mM to 100mM, 60mM to 100mM, 70mM to 100mM, 80mM to 100mM, 50mM to 150mM, 60mM to 150mM, 70mM to 150mM, 80mM to 150mM, 90mM to 150mM 100mM to 150mM, 10mM to 200mM, 15mM to 200mM, 20mM to 200mM, 25mM to 200mM, 30mM to 200mM, 40mM to 200mM, 50mM to 200mM, 60mM to 200mM, 70mM to 200mM, 80mM to 200mM, 90mM to 200mM, 100mM to 200mM, 150mM to 200mM 10mM to 250mM, 15mM to 250mM, 20mM to 250mM, 25mM to 250mM, 30mM to 250mM, 40mM to 250mM, 50mM to 250mM, 60mM to 250mM, 70mM to 250mM, 80mM to 250mM, 90mM to 250mM, 100mM to 250mM, 150mM to 250mM, or 200mM to 250mM. In another embodiment, the concentration of the buffer solution in the formulation is about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 90 mM, 95 mM, or about 100 mM. In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 20 mM. In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the buffer is present in the solution at a concentration of at least or equal to 50 mM. In embodiments where the solution is specifically reported to contain two or more (e.g., at least two, three, four, five, six, seven, eight, nine, or ten or more) different buffers, each of the two or more buffers may be present independently at, for example, one of the concentrations described above.

[0193] In one embodiment, the aqueous solution has a neutral pH or can be adjusted to have a neutral pH. As used herein, “neutral pH” is a pH between 7 and 8 (including the extreme values). Thus, as used herein, neutral pH includes specific pH values ​​such as 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, and 8.0. In some embodiments, the neutral pH is at least pH 7 (e.g., at least pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.7, or 7.9), but less than pH 8 (e.g., less than pH 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, or 7.1). That is, in some embodiments, the neutral pH can be, for example, at least pH 7, but less than pH 7.5. In some embodiments, the neutral pH can be between pH 7 and pH 7.5. In some embodiments, the neutral pH may be between pH 7 and pH 7.2. In another embodiment, the pH of the solution is between 7.0 and 7.4. In another embodiment, the pH of the solution is between 7.2 and 7.8. In yet another embodiment, the pH of the solution is between 7.2 and 7.6. In some embodiments, the neutral pH may be, for example, pH 7. Those skilled in the art will also recognize that human blood (such as blood from healthy patients) has a neutral pH as defined herein, for example, the pH of human blood is approximately pH 7.35 to pH 7.45. See, for example, Boron and Boulpaep (2003), “Medical physiology: acellular and molecular approach,” WBSaunders, New York (ISBN: 0721632564). In some embodiments, the pH of the highly concentrated antibody solution described herein is between approximately 6.4 and 7.5 (inclusive) (e.g., approximately 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, or 7.7). In one embodiment, the pH of the solution is between 7.2 and 7.6. In a particular embodiment, the pH of the solution is 7.4.

[0194] In one embodiment, the solution contains one or more surfactants, such as anionic, cationic, or nonionic surfactants. As used herein, the term "surfactant" refers to a surface-active molecule that contains both a hydrophobic portion (e.g., an alkyl chain) and a hydrophilic portion (e.g., a carboxyl group and a carboxyl group). Surfactants suitable for use in the formulations of this invention include, but are not limited to, fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, fatty acid glycerides (e.g., glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate), fatty acid polyglycerides (e.g., glyceryl monostearate, glyceryl distearate, glyceryl monolinoleate), and polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate). Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate), polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene fatty acid glycerides (e.g., polyoxyethylene monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., Polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitan beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene...). Ethylene stearamide; C12-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (with an average addition of 2 to 4 moles of ethylene oxide units) (e.g., sodium polyoxyethylene lauryl sulfate) and C10-C18 alkyl sulfonate succinates (e.g., sodium lauryl sulfonate succinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids.

[0195] In one embodiment, the surfactant in the formulation is a nonionic surfactant. In some embodiments, the surfactant in the formulation is a polyoxyethylene sorbitan fatty acid ester, such as polysorbate 20, 40, 60, 80, or a combination of one or more thereof. In one embodiment, the surfactant in the formulation is polysorbate 80 (Tween 80). In another embodiment, the surfactant in the formulation is polysorbate 60. In another embodiment, the surfactant in the formulation is polysorbate 40. In yet another embodiment, the surfactant in the formulation is polysorbate 20 (Tween 20).

[0196] The amount of surfactant added to the formulation is sufficient to reduce the aggregation of the formulated antibody and / or minimize the formation of particles in the formulation. For example, the surfactant may be present in the formulation in an amount of about 0.001% to about 1%, or about 0.001% to about 0.5%, or about 0.01% to about 0.2%. In one embodiment, the aqueous solution contains at least or about 0.001% (e.g., at least or about 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%). Surfactants of 0.19%, 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, or 0.5% or higher. In another embodiment, the aqueous solution contains no more than 0.2% (e.g., no more than 0.19%, 0.18%, 0.17%, 0.16%, 0.15%, 0.14%, 0.13%, 0.12%, 0.11%, 0.10%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, or 0.001%) of a pharmaceutically acceptable surfactant.

[0197] In another embodiment, the formulation comprises polysorbate at a concentration of about 0.001% to about 0.5%, about 0.005% to about 0.2%, about 0.01% to about 0.1%, or about 0.02% to about 0.06% or about 0.03% to about 0.05% (w / v). In some embodiments, the formulation comprises polysorbate at a concentration of 0.01% or 0.02% or 0.03% or 0.04% or 0.05% or 0.06% or 0.07% or 0.08% or 0.09% or 0.1% or 0.15% or 0.2% (w / v). In some embodiments, a surfactant is present in the formulation at an amount of 0.02% or about 0.04% (w / v). In one embodiment, a surfactant is present in the formulation at an amount of 0.05% (w / v).

[0198] In one embodiment, the formulation comprises at least about 0.01%, at least about 0.02%, at least about 0.05%, at least about 0.1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, or at least about 0.5% of polysorbate 80. In some embodiments, the formulation comprises about 0.01% to about 0.5%, about 0.01% to about 0.3%, about 0.001% to about 0.2%, about 0.02% to about 0.5%, about 0.02% to about 0.3%, about 0.02% to about 0.2%, about 0.05% to about 0.5%, about 0.05% to about 0.3%, about 0.05% to about 0.2%, about 0.075% to about 0.5%, about 0.075% to about 0.3%, or about 0.075% to about 0.2% of polysorbate 80. In another embodiment, the formulation comprises about 0.01%, about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, or about 0.5% of polysorbate 80. In one embodiment, the formulation comprises about 0.05% polysorbate 80. In one embodiment, the formulation comprises about 0.04% polysorbate 80. In one embodiment, the formulation comprises about 0.03% polysorbate 80. In one embodiment, the formulation comprises about 0.02% polysorbate 80. In one embodiment, the formulation comprises about 0.01% polysorbate 80.

[0199] In one embodiment, the aqueous solution contains one or more salts, such as sodium chloride, potassium chloride, or magnesium chloride. In some embodiments, the aqueous solution described herein contains a salt at a concentration of at least 10 mM (e.g., at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mM or higher). In some embodiments, the aqueous solution described herein may contain a concentration of less than or about 200 mM (e.g., less than or about 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 or 10 mM). In some embodiments, the aqueous solution described herein may contain salt concentrations of approximately 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 10 mM to 100 mM, 15 mM to 100 mM, 20 mM to 100 mM, 25 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 1 50mM, 15mM to 150mM, 20mM to 150mM, 25mM to 150mM, 30mM to 150mM, 40mM to 150mM, 50mM to 100mM, 60mM to 100mM, 70mM to 100mM, 80mM to 100mM, 50mM to 150mM, 60mM to 150mM, 70mM to 150mM, 80mM to 150mM, 90mM to 150mM 100mM to 150mM, 10mM to 200mM, 15mM to 200mM, 20mM to 200mM, 25mM to 200mM, 30mM to 200mM, 40mM to 200mM, 50mM to 200mM, 60mM to 200mM, 70mM to 200mM, 80mM to 200mM, 90mM to 200mM, 100mM to 200mM, 150mM to 200mM 10mM to 250mM, 15mM to 250mM, 20mM to 250mM, 25mM to 250mM, 30mM to 250mM, 40mM to 250mM, 50mM to 250mM, 60mM to 250mM, 70mM to 250mM, 80mM to 250mM, 90mM to 250mM, 100mM to 250mM, 150mM to 250mM, or 200mM to 250mM.In embodiments where the solution in the special report contains two or more (e.g., at least two, three, four, five, six, seven, eight, nine, or ten or more) different salts, each of the two or more salts may be present independently at, for example, one of the concentrations described above.

[0200] In one embodiment, the aqueous solution contains one or more carbohydrate excipients. Suitable carbohydrate excipients are, for example, Katakam and Banga (1995) J Pharm Pharmacol. 47(2) .: 103-107; Andya et al. (2003) AAPS PharmSci 5(2) The following descriptions are provided in Article 10; and in Shire (2009), “Current Trends in Monoclonal Antibody Development and Manufacturing,” Vol. 11, Springer, p. 354. Suitable carbohydrate excipients for use in the solutions described herein include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbitol; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as maltodextrin, dextran, and starch; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, and sorbitol. In one embodiment, the carbohydrate excipient is present in the solution specifically reported herein at a concentration of at least or about 0.5% (e.g., at least or about 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, 4.75%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10% or higher). In embodiments where the solution, as specifically reported, contains two or more (e.g., at least two, three, four, five, six, seven, eight, nine, or ten or more) different carbohydrate excipients, each excipient may be present independently at any of the concentrations described above.

[0201] In another embodiment, the stabilized aqueous solution comprises one or more stabilizers. Exemplary stabilizers include, but are not limited to, polyols, sugars (e.g., sucrose or trehalose), amino acids (e.g., arginine), amines, and salting-out salts. In one embodiment, the solution comprises at least one stabilizer at a concentration of 2-10% (inclusive). In one embodiment, the solution comprises 5% sucrose. In another embodiment, the solution comprises at least one or more stabilizers at a concentration of 10 mM to 50 mM (inclusive). In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 20 mM. In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 25 mM. In another embodiment, the stabilizer is present in the solution at a concentration of at least or equal to 50 mM. In another embodiment, the solution comprises 25 mM arginine.

[0202] In one embodiment, the solution described herein contains one or more preservatives.

[0203] As used herein, the term "preservative" refers to a pharmaceutical agent that reduces bacterial activity and may optionally be added to the formulations described herein. The addition of preservatives can, for example, facilitate the production of multipurpose (multi-dosage) formulations. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, wherein the alkyl group is a long-chain compound), and benzyl chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol, and benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methylparaben or propylparaben; catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0204] In one embodiment, the stable aqueous solution contains no more than five agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than four agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than three agents in addition to the anti-C5 antibody. In another embodiment, the stable aqueous solution contains no more than two agents in addition to the anti-C5 antibody. In yet another embodiment, the stable aqueous solution contains no more than one agent in addition to the anti-C5 antibody.

[0205] In one embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50 ± 15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5 ± 3% (e.g., 2%, 3%, 4%, 5%) 6%, 7% or 8% sucrose; and 25 ± 10 mM (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM) arginine; wherein the pH of the solution is 7.4 ± 0.5 (e.g. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0206] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50 ± 15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5 ± 3% (e.g., 2%, 3%, 4%, 5%) 6%, 7% or 8% sucrose; and 25 ± 10 mM (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM) arginine; wherein the pH of the solution is 7.4 ± 0.5 (e.g. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0207] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50±15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5±3% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, or 8%) sucrose; 25±10 mM (e.g., 15, 16, 17, ... Arginine (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM); and 0.05 ± 0.03% (e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08%) of polysorbate 80, wherein the pH of the solution is 7.4 ± 0.5 (e.g., 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0208] In another embodiment, the stable aqueous solution comprises: an anti-C5 antibody at a concentration of 100 ± 20 mg / mL (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 mg / mL), said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, and a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 19. Light chain CDR1 containing the amino acid sequence described in SEQ ID NO: 4, light chain CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and light chain CDR3 containing the amino acid sequence described in SEQ ID NO: 6; 50±15 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 mM) phosphate buffer; 5±3% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, or 8%) sucrose; 25±10 mM (e.g., 15, 16, 17, ... Arginine (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 mM); and 0.05 ± 0.03% (e.g., 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07% and 0.08%) of polysorbate 80, wherein the pH of the solution is 7.4 ± 0.5 (e.g., 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9).

[0209] In another embodiment, a stable aqueous solution (e.g., a sterile solution) is provided, wherein the solution comprises (a) an anti-C5 antibody (e.g., reveluzumab) at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine. In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody (e.g., reveluzumab) at a concentration of 100 mg / mL, (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

[0210] In another embodiment, the stable aqueous solution comprises (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine.

[0211] In another embodiment, the stable aqueous solution contains no more than three additional agents. In another embodiment, the stable aqueous solution contains no more than two additional agents. In yet another embodiment, the stable aqueous solution contains no more than one additional agent.

[0212] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, and (d) about 25 mM arginine.

[0213] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, (b) about 50 mM phosphate buffer, (c) about 5% sucrose, (d) about 0.05% polysorbate 80, and (e) about 25 mM arginine.

[0214] In one embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; and (d) about 25 mM arginine.

[0215] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine.

[0216] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0217] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; (d) 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0218] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) about 50 mM phosphate buffer; (c) about 5% sucrose; (d) about 0.05% polysorbate 80; and (e) about 25 mM arginine.

[0219] In another embodiment, the stable aqueous solution comprises: (a) an anti-C5 antibody at a concentration of 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; (b) 50 mM phosphate buffer; (c) 5% sucrose; (d) 0.05% polysorbate 80; and (e) 25 mM arginine.

[0220] IV. Methods for preparing highly concentrated antibody solutions

[0221] This document also provides a method for preparing a highly concentrated anti-C5 antibody solution. In one embodiment, a method is provided for generating a stable concentrated antibody solution comprising: 100 mg / mL anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose; and 25 mM arginine, the method comprising:

[0222] i) Provide a first aqueous solution comprising the anti-C5 antibody, the first aqueous solution having a first formulation and containing no more than 10 mg / mL of the anti-C5 antibody;

[0223] ii) Percolate the first aqueous solution into a formulation containing 50 mM phosphate buffer, 5% sucrose, and 25 mM arginine at pH 7.4 to produce a second aqueous solution, wherein the second aqueous solution has a second formulation due to percolation; and

[0224] iii) Concentrate the second aqueous solution to produce a stable concentrated antibody solution containing 100 mg / mL of the anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine.

[0225] In another embodiment, a method is provided for producing a stable concentrated antibody solution comprising 100 mg / mL anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, and 0.05% polysorbate 80, the method comprising:

[0226] i) Provide a first aqueous solution comprising the anti-C5 antibody, the first aqueous solution having a first formulation and containing no more than 10 mg / mL of the anti-C5 antibody;

[0227] ii) Percolate the first aqueous solution into a formulation containing 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, and 0.05% polysorbate 80 at pH 7.4, thereby producing a second aqueous solution, wherein the second aqueous solution has a second formulation due to percolation; and

[0228] iii) Concentrate the second aqueous solution to produce a stable concentrated antibody solution containing 100 mg / mL of the anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose, 25 mM arginine and 0.05% polysorbate 80.

[0229] V. Application route

[0230] The solutions described herein can be administered to patients using a variety of methods, depending in part on the route of administration. These routes can be parenteral, such as intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), intraocular injection, intra-articular injection, or intramuscular injection (IM). As used herein, “parenteral administration,” “via parenteral administration,” and other grammatically equivalent phrases refer to administration methods typically performed by injection, excluding enteral and topical administration, including but not limited to intravenous, intranasal, intraocular, pulmonary, intramuscular, intra-arterial, intrathecal, intracapsular, intra-articular, intracapsular, intraorbital, intracardiac, intradermal, intrapulmonary, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, carotid, and intrasternal injections and infusions.

[0231] In one particular embodiment, the solution is administered via subcutaneous injection. Subcutaneous administration can be accomplished using a device. This device can be a syringe, a pre-filled syringe, a disposable or reusable autoinjector, a pen syringe, a patch syringe, a wearable syringe, a portable syringe infusion pump with a subcutaneous infusion kit, or other device.

[0232] In one embodiment, the solution described herein is delivered to a subject via topical application. As used herein, "topical application" or "topical delivery" means delivery that does not rely on the transport of the composition or active agent (e.g., anti-C5 antibody) to its intended target tissue or site via the vascular system. After topical application near the target tissue or site, the solution or one or more components thereof may diffuse to the intended target tissue or site.

[0233] For example, the solution can be delivered by injection or by implanting a device containing the solution. The implant can be a porous, non-porous, or gel-like material, including membranes such as silicone rubber membranes or fibers. The implant can be configured to continuously or periodically release the solution into the subject. See, for example, U.S. Patent Application Publication No. 20080241223; U.S. Patent Nos. 5,501,856; 4,863,457; and 3,710,795; EP488401; and EP 430539, the disclosures of each of which are incorporated herein by reference in their entirety. The solutions described herein can be delivered to the subject by implantable devices based on, for example, diffusive, corrosive, or convection systems, such as osmotic pumps, biodegradable implants, electrodiffusion systems, electroosmotic systems, vapor pressure pumps, electrolytic pumps, effervescent pumps, piezoelectric pumps, erosion-based systems, or electromechanical systems.

[0234] In one embodiment, the solution described herein may be applied topically to a joint (e.g., an active joint). For example, in an embodiment where the condition is arthritis, a therapeutically appropriate solution may be applied directly to the joint (e.g., into the joint space) or near the joint. Examples of intra-articular joints to which the compositions described herein may be applied topically include, for example, the hip, knee, elbow, wrist, sternoclavicular joint, temporomandibular joint, carpal joint, tarsal joint, ankle, and any other joint experiencing arthritis symptoms. The compositions described herein may also be applied to bursae, such as the acromial bursa, biceps radial bursa, elbow radial bursa, deltoid bursa, infrapatellar bursa, ischial bursa, and any other bursa known in the medical field.

[0235] In another embodiment, the solution described herein may be applied topically to the eye. As used herein, the term "eye" refers to any and all anatomical tissues and structures associated with the eye. In one embodiment, the solution described herein is applied to the posterior chamber of the eye. In another embodiment, the solution described herein is applied intravitreally. In yet another embodiment, the solution described herein is applied transscleral.

[0236] In some embodiments, such as those for treating or preventing conditions like COPD or asthma, the solution described herein may be administered to a subject via the lungs. Lung delivery of the drug may be achieved by inhalation, and inhalation administration herein may be oral and / or nasal. In one embodiment, the solution as described herein may be administered to the lungs of a subject via a nebulizer. The nebulizer uses compressed air to deliver the compound in the form of a liquefied aerosol or nebulizer. The nebulizer may be, for example, a jet nebulizer (e.g., an air or liquid jet nebulizer) or an ultrasonic nebulizer. Other devices and methods of intrapulmonary administration are set forth in, for example, U.S. Patent Application Publications 20050271660 and 20090110679, the disclosures of which are incorporated herein by reference in their entirety.

[0237] In another embodiment, the solution described herein is present in a unit dosage form that may be particularly suitable for self-administration. Products formulated according to this disclosure may be contained in a container, typically such as a vial, cartridge, pre-filled syringe, or disposable pen. A metering device, such as that described in U.S. Patent No. 6,302,855, may also be used. The injection system may include a delivery pen as described in U.S. Patent No. 5,308,341. Pen-type devices most commonly used for self-administering insulin to diabetic patients are well known in the art. Such devices may include at least one injection needle (e.g., a 31-gauge needle of about 5 to 8 mm in length), typically pre-filled with one or more therapeutic unit doses of solution, and may be used to rapidly deliver the solution to the subject with minimal pain.

[0238] VI. Treatment

[0239] The solution can be used to treat a variety of diseases and conditions in human patients. In one embodiment, the solution can be used to treat complement-related conditions, including but not limited to: rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; atypical hemolytic uremic syndrome (aHUS); typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); paroxysmal nocturnal hemoglobinuria (PNH); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD)); hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; oligoimmune vasculitis; epidermolysis bullosa; recurrent abortion; and traumatic brain injury (see, for example, Holers (2008) Immunological Reviews). 223: 300-316 and Holers and Thurman (2004) Molecular Immunology 41 (147-152).

[0240] In another embodiment, complement-related diseases are complement-related vascular diseases, such as, but not limited to, diabetes-related vascular diseases (e.g., diabetes-related vascular diseases of the eye), central retinal vein occlusion, cardiovascular diseases, myocarditis, cerebrovascular diseases, peripheral (e.g., musculoskeletal) vascular diseases, renal vascular diseases, mesenteric / intestinal vascular diseases, angiogenesis of grafts and / or replants, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-related vasculitis, rheumatoid arthritis-related vasculitis, immune complex vasculitis, Goyan's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease (arteritis), venous air embolism (VGE), and restenosis after stent placement, coronary rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA) (see, for example, U.S. Patent Application Publication No. 20070172483).

[0241] Other complement-related conditions include, but are not limited to, myasthenia gravis, cold agglutinin disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, Guillain-Barré syndrome, Diagos' disease, graft rejection (e.g., transplant rejection), sepsis, burns (e.g., severe burns), systemic inflammatory sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), idiopathic thrombocytopenic purpura (ITP), Goodpasser syndrome, antiphospholipid syndrome (APS), catastrophic APS (CAPS), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and chronic inflammatory demyelinating neuropathy.

[0242] In another embodiment, the solution described herein can be used to treat thrombotic microangiopathy (TMA), such as TMA associated with complement-related conditions such as any complement-related condition described herein.

[0243] Complement-related disorders also include complement-related lung diseases, such as, but not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, alpha-1 antitrypsin deficiency, emphysema, bronchiectasis, obliterative bronchiolitis, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular disease.

[0244] In another embodiment, the solution described herein is applied to a subject to treat, prevent, or improve at least one symptom of complement-related inflammation (e.g., complement-related inflammatory aspects of complement-related disorders) in the subject. For example, the composition can be used to treat, prevent, and / or improve one or more symptoms associated with complement-related inflammation, such as graft rejection / graft-versus-host disease (GVHD), reperfusion injury (e.g., after cardiopulmonary bypass or tissue transplantation), and tissue damage following other forms of traumatic injury (e.g., burns (e.g., severe burns), blunt trauma, spinal cord injury, or frostbite). See, for example, Park et al. (1999), Anesth Analg. 99(1) : 42-48; Tofukuji et al. (1998) J Thorac Cardiovasc Surg 116(6) : 1060-1068; Schmid et al. (1997) Shock 8(2) : 119-124; and Bless et al. (1999) Am J Physiol 276(1) :L57-L63.

[0245] In another embodiment, complement-mediated disease is complement-mediated vascular disease, such as, but not limited to, cardiovascular disease, myocarditis, cerebrovascular disease, peripheral (e.g., musculoskeletal) vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, angiogenesis of grafts and / or replants, vasculitis, Henoch-Schönlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, organ or tissue transplantation, Goran's disease, capillary leak syndrome, dilated cardiomyopathy, diabetic vascular disease, thoracic and abdominal aortic aneurysm, Kawasaki disease (arteritis), venous air embolism (VGE), and restenosis after stent placement, coronary rotational atherectomy, and percutaneous transluminal coronary angioplasty (PTCA) (see, for example, U.S. Patent Application Publication No. 20070172483).

[0246] VII. Combination therapy

[0247] In one embodiment, the solution described herein is administered to a patient as a single therapy. In another embodiment, the solution is administered together with one or more adjunct agents and / or other therapies (e.g., those suitable for treating complement-related conditions). For example, combination therapy may include administration to a human patient of one or more adjunct agents (e.g., anticoagulants, antihypertensive drugs, or anti-inflammatory drugs (e.g., steroids)) that provide therapeutic benefit to the patient. In one embodiment, the solution described herein is administered together with anti-inflammatory agents (e.g., NSAIDs, corticosteroids, methotrexate, hydroxychloroquine, anti-TNF agents (such as etanercept and infliximab), B-cell depleting agents (such as rituximab), interleukin-1 antagonists, or T-cell co-stimulation blockers (such as abatacept)).

[0248] The adjunctive medications used to treat complement-related conditions in the subjects will vary depending on the specific condition being treated, but may include, but are not limited to, one or more antihypertensive drugs (e.g., angiotensin-converting enzyme inhibitors, labetalol, hydralazine, nifedipine, calcium channel blockers, nitroglycerin, or sodium nitroprussiate), anticoagulants, corticosteroids (e.g., prednisone), immunosuppressants (e.g., vincristine or cyclosporine A), anticoagulants (e.g., warfarin, aspirin, heparin, phenylindone, fondaparinux, idraparinux), and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran). (dabigatran), fibrinolytics (e.g., ancrod, α-aminocaproic acid, antifibrinolytic enzyme-α1, prostacyclin, and defibrotide), antihypertensives (e.g., labetalol, hydralazine, nifedipine, calcium channel blockers, nitroglycerin, or sodium nitroprusside), lipid-lowering agents (e.g., hydroxymethylglutaryl-CoA reductase inhibitors), antiepileptics (e.g., magnesium sulfate), antithrombotic agents (e.g., heparin, antithrombin, prostacyclin, or low-dose aspirin), sympathetic neuron mimics (e.g., salbutamol), antibiotics, deoxyribonuclease (e.g., ... Anticholinergic drugs, anti-IgE inhibitors (e.g., anti-IgE antibodies), corticosteroids, or nonsteroidal anti-inflammatory drugs (NSAIDs) are all available. Many different NSAIDs are available, some of which are over-the-counter, including ibuprofen. and naproxen Many other medications are available by prescription, including meloxicam. Etodolac Nabumetone Sulindac Tolementin Magnesium choline salicylate Diclofenac Diflusinal Indomethacin Ketoprofen Oxaprozin And piroxicam (See, for example, Mihu et al. (2007) J Gastrointestin Liver Dis 16(4) (419-424). In another embodiment, the solution described herein can be formulated for administration to a patient in conjunction with intravenous gamma globulin therapy (IVIG), plasma ablation, plasma exchange, or plasma exchange.

[0249] In one embodiment, the solution is administered simultaneously with one or more additional drugs and / or therapies. In another embodiment, the solution is administered before the administration of one or more additional drugs and / or therapies. In yet another embodiment, the solution is administered after the administration of one or more additional drugs and / or therapies.

[0250] When the antibody solution described herein is used in combination with a second active agent, the agent (e.g., an anti-C5 antibody and a second agent) can be prepared separately or together. For example, the solution and agent can be mixed just before administration and administered together, or administered separately, for example, at the same or different times.

[0251] VIII. reagent kits and unit dosage forms

[0252] This document also provides a kit comprising a stable aqueous solution containing a therapeutically effective amount of an anti-C5 antibody or its antigen-binding fragment, such as revozumab or BNJ421, suitable for administration to human patients (e.g., patients with complement-related conditions). The kit may optionally include instructions for use, for example, including an administration schedule to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition contained therein to a patient.

[0253] The kit may also contain a suitable tool for delivering one or more solutions to a patient in need (e.g., a patient with, suspected of having, or at risk of developing complement-related conditions). In one embodiment, the tool is adapted to deliver the solution invasively (e.g., intravascularly, subcutaneously, intra-articularly, intraocularly, intravitreally, or intramuscularly) to the patient. In another embodiment, the tool is adapted to deliver the solution subcutaneously to the patient. In yet another embodiment, the tool is adapted to deliver the solution invasively to the patient. For example, the tool may be a syringe or an osmotic pump. In another embodiment, the solution may be formulated as eye drops, and the tool is a dropper.

[0254] Optionally, the kit includes multiple packets of single-dose solution, each containing an effective amount of solution for a single administration. The kit may also include the necessary instruments or devices for administering the solution. For example, the kit may provide one or more pre-filled syringes containing the solution.

[0255] The following embodiments are merely illustrative and should not be construed in any way as limiting the scope of this disclosure, as many variations and equivalents will become apparent to those skilled in the art upon reading this disclosure.

[0256] All references, Genbank entries, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0257] Example

[0258] Example 1: Development of a high-concentration formulation of revorutinumab (AXLN1210) for subcutaneous administration

[0259] This example summarizes the development of a high-concentration formulation of ALXN1210 for subcutaneous administration (e.g., 50 mM phosphate buffer, 5% sucrose, 25 mM arginine, pH 7.4 at 100 mg / mL). Preliminary experiments were conducted in early formulation development to obtain pre-formulation screening data and evaluate the reduction in the milky appearance of ALXN1210 at higher concentrations. The initial formulation of ALXN1210 (10 mM phosphate, 150 mM sodium chloride (pH 7.0), 0.02% Tween 80 at 10 mg / mL) was colorless and slightly milky. The milky appearance increased with increasing ALXN1210 concentration. Based on the results of the pre-formulation screening, stability studies were performed to obtain lead stability data. Following the initial stability studies, prototype stability studies were performed to obtain optimal formulations for the bulk active pharmaceutical ingredient and the pharmaceutical product. The preliminary studies and stability studies are discussed in detail below.

[0260] 1. method

[0261] A. Appearance

[0262] The appearance was determined by visual observation against white and black backgrounds using normal laboratory light.

[0263] B. C5 combination

[0264] C5 binding ELISA is a efficacy assay for ALXN1210. This assay is a direct binding immunoassay with colorimetric detection, used to test the ability of ALXN1210 to bind to its target, human C5 complement protein. Polysorp microtiter plates were coated with human C5 protein and blocked with bovine serum albumin (BSA). A standard curve was prepared using ALXN1210 reference material. Reference material and test samples were prepared at three dilutions designed to fall within the working range of the assay. After incubation with standards and samples, the plates were washed and incubated with mouse anti-human IgG4 conjugated with horseradish peroxidase (HRP). The plates were washed again and then developed with substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The amount of substrate in the reaction was read spectrophotometrically at 405 nm on a microplate reader. The absorbance readings were proportional to the concentration of ALXN1210 bound to C5 on the plate. A four-parameter curve was fitted to the standard curve, and the results for the reference material and test samples were obtained from the curve using interpolation. The test results for the samples were compared with those for the reference material, and the relative activity (%) was reported.

[0265] C. density

[0266] Density measurements using the DMA 4500 densitometer are determined using the U-tube principle. A hollow U-shaped glass tube is filled with the sample, and then electron excitation is performed with the lowest possible amplitude. The density is determined through the following relationship:

[0267] ρ=A(τ 2 )-B

[0268] p = density

[0269] τ = Oscillation period

[0270] A and B are instrument constants determined by instrument calibration using two substances of known density.

[0271] D. Differential scanning fluorescence method

[0272] Differential scanning fluorescence (DSF) measures changes in thermal stability by completing a thermal denaturation curve in the presence of a fluorescent dye (such as Sypro Orange). When a protein unfolds, the exposed hydrophobic surface binds to the dye, thereby increasing fluorescence and exposing the T1 surface to the hydrophobic layer. h A stability curve with a characteristic midpoint value is generated at a certain temperature.

[0273] E. Dynamic light scattering method

[0274] Dynamic light scattering (DLS) measures the size and interactions of in-situ proteins, nanoparticles, and other macromolecules in microplates by using an illumination system that allows imaging of the wells in the microplate with a 3-megapixel airborne camera. The fluctuations in light scattering due to Brownian motion give the diffusion coefficient, which is related to the hydrodynamic radius of the particles present in solution.

[0275] F. HPLC gel permeation method

[0276] Gel permeation (size exclusion) HPLC is used to distinguish monomeric IgG from larger multimeric antibody substances that may result from monomer aggregation. The test sample is injected onto a TSK G3000 SWXL column equilibrated with phosphate-buffered saline (pH 7.0) and then isocratically eluted. The protein peak is monitored at 214 nm, and the purity percentage of monomeric IgG is expressed as a percentage of the total integrated peak area. Larger multimers are detected by observing the peak eluted before the monomer peak.

[0277] G. Imaging capillary electrophoresis (iCE)

[0278] This method uses the Protein Simple iCE280 or iCE3 system, which performs free solution IEF in a capillary column and uses a whole-column UV detector to detect the focused protein region. Samples are prepared by pre-mixing ALXN1210, a carrier amphoteric electrolyte, and pI markers. Samples are loaded into a capillary cassette, and acid and base are added to the electrolytic cells at both ends of the capillary. A voltage is applied to focus the analyte at its pI. A CCD camera captures one UV absorption image of the entire capillary column every 30 seconds, allowing for real-time monitoring of the focusing process. The resulting separation patterns are captured and analyzed. The pI of the proteins present in the sample is obtained by interpolation from the location of the pI marker incorporated into the sample.

[0279] H. Lab-on-a-Chip (LoC)

[0280] This method tests the homogeneity and purity of the product. Non-reduced samples are denatured by treatment with lithium dodecyl sulfate (LDS). Reduced samples are denatured by treatment with lithium dodecyl sulfate (LDS) and disulfide bonds are broken with dithiothreitol (DTT). Peptide chains are mixed with fluorescent dyes bound to LDS and separated according to molecular size by microcapillary electrophoresis. Proteins are detected and quantified by laser-induced fluorescence.

[0281] I. molar osmotic concentration

[0282] The molar osmolality of the sample was determined using a freezing point depression osmoremeter. Before use, the osmoremeter was calibrated using commercially available, verified molar osmolality standards of 50 mOsm / kg and 850 mOsm / kg, covering the sample range. Calibration was confirmed using a 290 mOsm / kg reference solution before testing the sample. Three copies of the sample were tested, and the average value of the measured values ​​was reported.

[0283] J. pH

[0284] pH measurements were performed using a protein-resistant saturated silver-free KCl combination electrode, along with associated instruments and a temperature monitor. The instruments were calibrated prior to use with commercially available solutions within the appropriate pH range (i.e., pH 4.0–pH 7.0).

[0285] K. Protein concentration using SoloVPE

[0286] Using the theoretically determined extinction coefficient of 1.479, protein concentration was determined in the test samples using a variable optical path technique and absorbance at 280 nm. For each sample, three replicate absorbance readings were performed according to this method.

[0287] L. Viscosity

[0288] Viscosity measurements obtained using the AMVn viscometer are determined via the rolling ball principle. A sample and a solid ball of known density are placed in a hollow tube, which is then tilted at a known angle. The time required for the ball to move from one side of the tube to the other is measured and used to calculate the viscosity using the following formula:

[0289] η=K*(ρb-ρs)*tr

[0290] η = Dynamic viscosity (mPa*s)

[0291] K = proportionality constant

[0292] ρb = density of the sphere (g / mL)

[0293] ps = Sample density (g / mL)

[0294] tr = ball rolling time

[0295] To calculate viscosity, the sample density measured using a DMA 4500M densitometer was used as ps.

[0296] M. Determination of invisible particles by microfluidic imaging (MGI)

[0297] The objective was to evaluate all invisible particles in the formulation using microfluidic imaging (MFI). Samples were removed from storage conditions of 2–8°C and tested directly in the MFI using the BOT1 autosampler. Six sample inversions were performed before loading the samples into the BOT1 to ensure complete particle mixing. Samples were loaded into three consecutive wells, with one measurement performed in each well, for a total of three repetitions. Three mixing cycles were established within the BOT1 to further ensure homogeneous mixing.

[0298] 2. Formulation development

[0299] Figure 1-5 Tables 1-5 show the experimental results of early formulation development of high-concentration ALXN1210.

[0300] In the first experiment, the effect of adding amino acids to ALXN1210 in sodium phosphate buffer on emulsion turbidity was observed. The cause of the turbidity was determined to be the lack of charge repulsion between antibody molecules in the solution at high concentrations. A series of experiments were performed as described below to optimize the specific amino acids and concentrations required to produce a stable, clear solution. Based on these experiments, it was determined that the addition of a positively charged amino acid (L-arginine) reduced the emulsion turbidity of a 50 mg / mL ALXN1210 sample in sodium phosphate buffer. Visual inspection of the vials yielded the same conclusion (data not shown).

[0301] In addition, the following experiments were performed using 10 mg / mL ALXN1210 IV formulation and antibody concentration, followed by buffer exchange to evaluate various initial buffer systems for discovering high concentrations of ALXN1210. As shown in Table 1 below, a final buffer exchange ratio of 1:1000 was required for all pooled samples to obtain the desired pH. The concentration range of the pooled samples was 35.3 to 54.0 mg / mL. The recoveries after buffer exchange ranged from 70.6% to 108%. Visual results showed that vials exchanged with 25 mM histidine and 25 mM phosphate buffer at pH 7 were clear and colorless, comparable to eculizumab, while solutions in all vials exchanged with other buffers were opaque. Imaging capillary electrophoresis (iCE) results showed pIs ranging from 5.98 to 6.54, major pIs ranging from 6.19 to 6.24, and area percentages ranging from 63.1% to 65.9%. Size exclusion chromatography (SEC) results showed that the monomer percentage (purity) was 98.48% to 98.98%.

[0302] Table 1: Buffer exchange of ALXN1210 from 10 mg / mL to 50 mg / mL (pooled groups 1-3)

[0303]

[0304] *Comparable to icurizumab

[0305] like Figure 1 As shown, the results of salt titration of samples exchanged with histidine buffer using DLS at pH 7 showed that self-association in ALXN1210 increased with increasing salt concentration.

[0306] like Figure 2 As shown, the results of L-arginine titration using dynamic light scattering (DLS) indicate that 25 mM L-arginine is the minimum amount required to reduce the turbidity in 50 mg / mL ALMN1210.

[0307] like Figure 3 As shown, the salt titration results of the phosphate-buffered buffer-exchanged samples using DLS at pH 7 show that in ALXN1210, both salt-free and with the addition of 150 mM salt exhibit minimal self-association. Peaks labeled 2 and 5 are compared.

[0308] like Figure 4 As shown, the results of buffer exchange samples obtained using DSF indicate that the hydrophobic pocket was not exposed in the ALXN1210. Citrate and acetate buffers at pH 5 and 6 exhibited low thermal stability and the lowest melting temperature (Tm), while histidine and phosphate buffers at pH 7 were the most stable and had the highest Tm.

[0309] As shown in Table 2, ALXN1210 was clear and colorless when 25 mM L-arginine was added to 25 mM phosphate buffer at pH 7, at a concentration of approximately 100 mg / mL.

[0310] Table 2: Appearance of ALXN1210 100 mg / mL sample

[0311]

[0312] like Figure 5 As shown, the results of 10 mg / mL and 114 mg / mL ALXN1210 obtained using DLS with and without L-arginine incorporation indicate that adding 25 mM L-arginine to a sample of at least 100 mg / mL is very comparable to the 10 mg / mL reference. The sample of at least 100 mg / mL without added L-arginine produced a more advanced substance, indicating the presence of self-association.

[0313] The results of the molar osmolality concentrations presented in Table 3 show that ALXN1210 in 25 mM histidine at pH 7.2 containing 8% sucrose or 4.5% sorbitol falls within the desired molar osmolality concentration range of 275-320. The molar osmolality concentrations of ALXN1210 in 25 mM phosphate and 25 mM L-arginine at pH 7 supplemented with 7% sucrose or 4% sorbitol also fall within the desired range.

[0314] Table 3: Molar osmotic concentration of ALXN1210 in various formulations

[0315]

[0316]

[0317] The viscosity results presented in Table 4 show that the viscosity of ALXN1210 solutions in histidine and phosphate buffer increases with increasing ALXN1210 concentration. The density results presented in Table 4 show that the density of histidine and phosphate buffer does not change significantly with varying concentrations.

[0318] Table 4: Viscosity and density of ALXN1210 samples at different concentrations

[0319]

[0320]

[0321] As shown in Table 5, the addition of L-arginine base significantly increased the pH of the samples. L-arginine QS containing sodium dihydrogen phosphate added to the samples increased the pH by 1 pH unit. L-arginine HCl added to the samples decreased the pH by approximately 0.25 pH units. However, the appearance showed a decrease in turbidity from L-arginine HCl added to L-arginine QS added to L-arginine base added.

[0322] Table 5: pH of L-arginine buffer solution used for 25 mM L-arginine infusion and its effect on the appearance of ALXN1210

[0323]

[0324]

[0325] 3. Long-term stability

[0326] Figure 6-21 The results of the initial stability studies are presented. These results show that after 2 months at 2–8°C, 23–27°C, and 37°C, the histidine formulation was the least stable, while the phosphate formulation was the most stable. Similarly, as demonstrated by size exclusion chromatography, after 2 months at 2–8°C and 23–27°C, sorbitol and sucrose were comparable in the phosphate formulation. However, after 2 months at 37°C, sorbitol was slightly more stable than sucrose. Dynamic light scattering (DLS) results showed no significant changes in the phosphate sample containing 25 mM L-arginine after the addition of sucrose or sorbitol, after 2 months at 2–8°C. The DLS results for the histidine sample at T=2 months were not superimposed due to high polydispersity during the sampling interval, indicating that the stability of this formulation was worse than that of the phosphate formulation. The 5-day freeze-thaw cycle results showed no significant changes between T=0 and 5 freeze-thaw cycles.

[0327] 4. Prototype Formula

[0328] Figure 22-46 The results of the stability prototype study are shown.

[0329] These results show that all phosphate formulations (bulk active pharmaceutical ingredient (BDS) and pharmaceutical product (DP)) at 75 mg / mL and 100 mg / mL were stable during stability studies at 2–8 °C, -20 °C, and -80 °C. After five freeze-thaw cycles at -20 °C and -80 °C, all 100 mg / mL bulk active pharmaceutical ingredient formulations were stable and showed no significant changes.

[0330] 5. Intermediate conclusions prior to short-term degradation test

[0331] Based on the results of these studies, the optimal formulation for high-concentration ALXN1210 was determined. Preliminary experiments showed that the addition of L-arginine reduced the milky appearance of 100 mg / mL ALXN1210. Initial stability studies led to the selection of a pilot formulation containing >50 mg / mL L-arginine in phosphate buffer. Prototype stability studies determined that the initial optimal formulation for ALXN1210 at 100 mg / mL was 50 mg phosphate buffer, 5% sucrose, 25 mM arginine, and pH 7.4.

[0332] 6. Development of the final optimal formula

[0333] A short-term degradation study was conducted to assess the suitability of the initial optimal formulation (100 mg / mL ALXN1210) in a preparation buffer (50 mM sodium phosphate, 25 mM arginine, and 5% sucrose, pH 7.4) to evaluate whether polysorbate 80 (PS80) or other surfactants were necessary to prevent degradation. Two brands of PS80 are reportedly 0.05% (w / v) NOF from America Corporation POLYSORBATE 80 (HX2), composed of >99% pure oleic acid. TM and AVANTOR TM 4117J.T. Polysorbate 80 is a widely used surfactant composed of a blend of fatty acids, including oleic and palmitic acids. Both products are often referred to as TWEEN. It is a nonionic surfactant derived from polyethoxylated sorbitan anhydride and oleic acid, and has hydrophilic groups derived from ethylene oxide polymers.

[0334] Table 6 below lists the test methods used to evaluate the potential use of JTbaker Avantor 4117PS80 in a 100 mg / mL ALXN1210 formulation. Please refer to the individual test methods for more detailed information and method descriptions.

[0335] Table 6: Test Methods for Potential Uses of T. Baker Avantor 4117PS80

[0336]

[0337]

[0338] Visual inspection was performed on vials containing 100 mg / mL ALXN1210 and JTBaker Avantor 4117PS80 or HX2 NOFPS80. No visible particles or significant color changes were observed in any of the samples exposed to degradation storage conditions at 45°C and then stirred at 2–8°C for 5 days. The results are shown in Table 7.

[0339] Table 7. Visual inspection of 100 mg / mL ALXN1210 in 5 cc vials containing JT Baker Avantor 4117 PS80 or HX2 NOF PS80

[0340]

[0341]

[0342] The ALXN12 concentration of 10 mg / mL shows a slight decrease due to degradation conditions. See Table 8 for all concentration measurements.

[0343] like Figure 2 As shown, when exposed to the same degradation conditions, the concentration of ALXN1210 100 mg / mL formulation containing 0.05% Avantor PS80 or HX2 NOF PS80 did not change significantly.

[0344] Table 8: Concentration Measurements

[0345]

[0346] As shown in Table 8 above, when exposed to the same degradation conditions, the concentration of ALXN1210 100 mg / mL formulations containing 0.05% Avantor PS80 or HX2 NOF PS80 did not change significantly. The concentrations remained comparable among the ALXN1210 100 mg / mL formulations containing 0.05% Avantor PS80 or HX2 NOF PS80.

[0347] Turbidity was measured by monitoring absorbance at 650 nm. The measured values ​​are shown in Table 9.

[0348] The ALXN1210 100 mg / mL formulation containing 0.05% Avantor PS80 or 0.05% HX2 NOF PS80 showed no significant change in turbidity. In this study, turbidity remained stable at all time points and under all degradation conditions.

[0349] Table 9: Turbidity measured by monitoring absorbance at 650 nm

[0350]

[0351]

[0352] For samples incubated at 45°C for 7 or 14 days, followed by shaking (200 RPM) at 2–8°C, a decrease in monomer percentage was observed as expected due to degradation conditions. However, when exposed to the same time points and conditions, no substantial difference in monomer percentage was observed between the two ALXN1210 100 mg / mL formulations containing 0.05% Avantor PS80 or 0.05% HX2 NOF PS80.

[0353] The percentage of individual data is shown in Table 10. Figure 4 The results showed that the monomer percentage decreased as expected after degradation conditions, and there was no significant difference between the two ALXN1210 100 mg / mL formulations containing 0.05% Avantor PS80 or 0.05% HX2 NOF PS80 when exposed to the same time points and conditions.

[0354] Table 10: Monomer percentage of samples incubated at 45°C for 7 or 14 days, followed by shaking at 2-8°C (200 RPM).

[0355]

[0356]

[0357] After incubating ALXN1210 with 0.05% JTBaker Avantor polysorbate 80 or 0.05% HX2 NOF polysorbate 80 at 45°C for 7 and 14 days, respectively, the transition to acidic substances was detected by isoelectric point focusing, as shown in Table 11. Further shaking of the sample had no significant effect on the further transition of the main peak to acidic substances.

[0358] Table 11: Isoelectric point focusing via CE-SDS

[0359]

[0360] A vibration stress test was performed on the initial optimal formulation (100 mg / mL ALXN1210 preparation buffer containing 50 mM sodium phosphate, 25 mM arginine, and 5% sucrose) at pH 7.4, with and without PS 80 from two brands at a concentration of 0.05%. The formation of invisible particles was used to assess degradation. Samples were shaken at 200 rpm at a temperature between 2–8 °C. The presence of invisible particle formation was measured at 0, 1, 3, and 5 days. The results, as shown in Table 12, indicate that the addition of 0.05% PS80 to the formulation significantly reduced the formation of invisible particles when high-concentration formulations were subjected to short-term stresses, such as 200 rpm vibration.

[0361] Table 12: Reducing the appearance of invisible particles by adding PS80 and filtration

[0362]

[0363]

[0364] 7. Conclusion

[0365] In summary, the optimal subcutaneous formulation of ALXN1210 at 100 mg / ml is a buffer solution containing 50 mM sodium phosphate, 25 mM arginine, and 5% sucrose, along with 0.05% PS80 (pH 7.4).

[0366] Example 2: A Phase 1 study evaluating a single subcutaneous dose of ALXN1210 versus intravenous administration in healthy subjects.

[0367] A phase 1 study was conducted to evaluate the safety, tolerability, pharmacokinetic (PK) / pharmacodynamic (PD) efficacy, and immunogenicity of subcutaneous (SC) administration of antibody BNJ441 (also known as ALXN1210) compared to intravenous (IV) administration in healthy subjects.

[0368] 1. Purpose

[0369] The main objectives of this study were (1) to evaluate the safety and tolerability of a single subcutaneous dose of ALXN1210 compared with a single intravenous dose of ALXN1210 in healthy subjects, as assessed by physical examination results, vital sign measurements, immunogenicity, laboratory analysis and assessment of adverse events (AEs), and (2) to determine the absolute bioavailability of the subcutaneous ALXN1210.

[0370] The secondary objective was to evaluate the PD effect of subcutaneously administered ALXN1210 compared to intravenously administered ALXN1210, as assessed by free C5 and chicken erythrocyte (cRBC) hemolysis levels.

[0371] 2. Research Design

[0372] Overall research design such as Figure 47 As described, this was conducted as a Phase 1 study designed to evaluate the safety, tolerability, pharmacokinetics, PD, and immunogenicity of a single subcutaneous dose of 400 mg ALXN1210 compared to a single intravenous dose of 400 mg ALXN1210 or a subcutaneous placebo in 42 healthy subjects. Eligibility was screened for all subjects. Subjects who did not meet the eligibility criteria were not rescreened for the study unless the ineligible condition was transient, self-limiting, easily treatable, and expected to be resolved at the time of administration.

[0373] Initially, six subjects were randomly assigned in a blinded 2:1 ratio to cohort 1a to receive either a single subcutaneous dose of 400 mg ALXN1210 or a single subcutaneous dose of placebo. Clinical safety data for the first 48 hours after dosing were evaluated in subjects in cohort 1a before recruitment to cohort 1b or 2 began. Thirty-six subjects were then randomly assigned in a 2:1 ratio to either cohort 1b (N=24) or cohort 2 (N=12). In cohort 1b, 24 subjects were further randomly assigned in a 5:1 ratio and blinded to receive either a single subcutaneous dose of 400 mg ALXN1210 (20 subjects) or a single subcutaneous dose of placebo (4 subjects). Twelve subjects in cohort 2 received a single intravenous dose of 400 mg ALXN1210 in an open-label manner.

[0374] As appropriate, all recruited participants were included in the analysis. Participants from cohorts 1a and 1b were combined for analysis. Participants attended the study for up to 39 weeks, including a 70-day screening period followed by a 200-day follow-up period for safety, pharmacokinetic, PD, and immunogenicity assessments after administration of the study drug.

[0375] In this study, 42 participants were evaluated for primary and secondary objectives: 6 participants in group 1a (4 receiving ALXN1210 subcutaneously and 2 receiving placebo subcutaneously); 24 participants in group 1b (20 receiving ALXN1210 subcutaneously and 4 receiving placebo subcutaneously); and 12 participants in group 2 (ALXN1210 IV).

[0376] 3. Dosage principles

[0377] A single subcutaneous dose of 400 mg (equivalent to 4 mL) is administered via injection into the abdominal region. Acceptable safety is anticipated with a single 400 mg dose of ALXN1210 SC. The administration of a single 400 mg dose of ALXN1210 SC and placebo SC as described in this protocol is expected to provide data from which multi-dose simulations can be derived to design the dosing regimen required to achieve therapeutic serum concentrations (>50 μg / mL) in patients.

[0378] Based on a review of clinical safety data from 6 subjects in group 1a within the first 48 hours after administration, 36 subjects were randomly assigned in parallel to groups 1b and 2. Recruitment to groups 1b and 2 was conducted as described in Table 13.

[0379] The group toxicity rules are as follows. Toxicity refers to clinically significant drug-related adverse reactions. "Group progression" refers to the progression of a continuous dose / dosing regimen that meets the dose progression rules and minimum data requirements. "Pause" means that IMP is no longer administered at the relevant dose level / dosing regimen, and further group progression is paused.

[0380] Table 13: Toxicity Rules

[0381]

[0382]

[0383] Abbreviations: CTCAE = Standard Terminology for Commonly Used Adverse Events; SAE = Serious Adverse Events; SOC = System Organ Classification.

[0384] 4. Evaluation Timeline

[0385] Table 14-15 provides the timeline for the research procedures used.

[0386] Table 14: Assessment Timeline: Screening to First Visit

[0387]

[0388]

[0389] The permissible research evaluation window is described in the Research Operations Manual.

[0390] 2. End of Infusion (EOI) occurs approximately 15 minutes after Start of Infusion (SOI).

[0391] 3. After completing all assessments on day 5, participants left the clinical research unit. Participants were provided with a "Research Participant ID Card" containing information about the healthcare provider and the participant's symptoms of meningitis infection.

[0392] 4. Obtain a signed and dated informed consent form before conducting any study-specific screening procedures.

[0393] 5. For subjects with insufficient prior MCV4 immunization or serogroup B vaccination records, MCV4 immunization should be performed at least 56 days before the first dose on day 1, and vaccination against serogroup B meningococcal infection should be administered at least 56 days before administration on day 1, with a booster dose administered at least 28 days before administration on day 1.

[0394] 6. Complement activity should be confirmed during screening (by appropriate assays such as complement alternative pathway (CAP) ELISA / C5 (hemolysis) inhibition) to confirm that the subject does not have complement deficiency.

[0395] 7. If the sample taken after administration indicates that complement has not been normalized, store the sample taken on day -1 for future analysis.

[0396] 8. Serum pregnancy tests were performed on all female subjects to confirm that they were not pregnant.

[0397] 9. On day 1, vital signs measurements were assessed before administration (within 15 minutes before SOI), at the end of infusion, 30 minutes after the end of infusion, 2 hours after the start of infusion, 4 hours after the start of infusion, and 8 hours after the start of infusion.

[0398] 10 On day 1, three 12-lead ECGs were obtained before administration and approximately 15 minutes after the infusion.

[0399] Continuous cardiac recordings were performed before administration and during the duration of IV infusion (Group 2), and before administration until 3 hours after SC injection (Groups 1a and 1b).

[0400] 12. Evaluate the infusion or injection site within 15 minutes of starting the infusion / injection and within ±15 minutes of other scheduled times on Day 1. Indurations or reactions <1 cm are not considered adverse events unless they persist for more than 24 hours. Assess pain at the infusion or injection site using a visual analog scale (0-10). No pain assessment was performed prior to administration.

[0401] 13 At each visit, the researcher or designated person meets with the subject to discuss the potential safety risks of ALXN1210 and address any safety concerns for the subject.

[0402] 14. Begin collecting adverse events (AEs) and serious adverse events (SAEs) from the time the informed consent form is signed.

[0403] 15. Prophylactic antibiotic treatment was administered to the subjects starting at night of day -1, with oral penicillin V 500 mg twice daily (equivalent to 1 × 10⁶ units) until complement activity was normalized by CH₅₀ assay.

[0404] Abbreviations: ADA = Anti-drug antibody; BMI = Body mass index; cRBC = Chicken red blood cells; CRU = Clinical research unit; ECG = Electrocardiogram; EOI = End of infusion / injection; HIV = Human immunodeficiency virus; ICF = Informed consent form; MCV4 = Quadrivalent meningococcal vaccine; OP = Outpatient clinic; SOI = Start of infusion / injection; TB = Tuberculosis

[0405] Table 15: Assessment Schedule: From the 2nd to the 14th Visit

[0406]

[0407] 1. Collect other samples after day 57.

[0408] 2. At each visit, the researcher or designated person met with the subject to discuss the potential safety risks of ALXN1210 and address any safety concerns for the subject.

[0409] 3. Begin collecting adverse events from the time the informed consent form is signed.

[0410] 4. Subjects were given prophylactic antibiotic treatment, including oral penicillin V 500 mg twice daily (equivalent to 1×10⁻⁶ mg). 6 (units), until complement activity is normalized by CH50 assay.

[0411] Abbreviations: ADA = Anti-drug antibody; cRBC = Chicken red blood cell; CRU = Clinical research unit; ECG = Electrocardiogram; ICF = Informed consent form; OP = Outpatient clinic

[0412] 5. Subject selection and withdrawal

[0413] Participants must meet all of the following criteria to be eligible for the study:

[0414] 1. Healthy subjects aged 25 to 55 years (inclusive) at the time of administration.

[0415] 2. Body Mass Index (BMI) between 18 and 29.9 kg / m² 2 (Including the extreme values), and the weight is 50 to 100 kg (inclusive).

[0416] 3. At screening and before administration on day 1, the QT interval was corrected for ≤450 ms for men and ≤470 ms for women using the Fridericia formula (QTcF).

[0417] 4. Willing and able to provide written informed consent and adhere to the study's schedule.

[0418] 5. Documented MCV4 vaccination must have been administered at least 56 days prior to drug administration and no more than 3 years prior. Records must include a positive antibody titer to confirm an immune response prior to administration of the investigational drug.

[0419] 6. Administer serogroup B meningococcal vaccine at least 56 days before day 1 administration, administer a booster at least 28 days before day 1 administration, and the interval between the first and second injections should be at least 28 days.

[0420] 7. Female subjects of fertility who are sexually active with the opposite sex must use a highly effective or acceptable method of contraception as defined above, from the start of screening and for at least 6 months after administration of the study drug. Antibiotic prophylaxis is required in this study, which may impair the effectiveness of hormonal contraception. Therefore, it is recommended that subjects using hormonal contraception also use barrier contraception (e.g., condoms or diaphragms containing spermicide) during the duration of hormonal prophylaxis. Male subjects who are sexually active with the opposite sex and have a fertile female partner or spouse, or a pregnant or breastfeeding partner, must consent to the use of barrier contraception (male condoms) during treatment and for at least 6 months after administration of the study drug. Barrier contraception is required even if a medical evaluation of a successful vasectomy is documented. Female partners of fertile male subjects must use a highly effective method of contraception as defined above, or an acceptable method of contraception as defined below, from the start of screening and for at least 6 months after administration of the study drug. Male subjects must not donate sperm during screening and treatment and for at least 6 months after administration of the study drug.

[0421] Subjects who meet any of the following exclusion criteria are not eligible to participate in this study:

[0422] 1. Subjects who have had close, prolonged contact with people under 2 years of age or over 65 years of age (defined as living under the same roof or providing personal care to them), or subjects who are immunocompromised or have one of the following underlying medical conditions: anatomical or functional asplenia (including sickle cell disease); congenital deficiency of complement, properin, factor D, or primary antibody; acquired complement deficiency (e.g., subjects receiving eculizumab); or human immunodeficiency virus (HIV).

[0423] 2. Subjects who are one of the following: professionals exposed to environments with a high risk of meningococcal disease; research, industrial, and clinical laboratory personnel who are frequently exposed to Neisseria meningitidis; military personnel undergoing basic training (military personnel may have a higher risk of meningococcal infection when living in close proximity); daycare center staff; people living on college or university campuses; and those who plan to travel to areas endemic for meningococcal meningitis (e.g., India, sub-Saharan Africa, or a pilgrimage to Saudi Arabia) during the study or who have already traveled within 6 months prior to administration.

[0424] 3. Any history of Neisseria infection.

[0425] 4. Unexplained history of recurrent infections, or a history of infection requiring treatment with systemic antibiotics within 90 days prior to administration.

[0426] 5. HIV infection (proven by HIV-1 or HIV-2 antibody titer).

[0427] 6. Acute or chronic hepatitis B virus (HBV) infection. All participants must undergo a hepatitis B surface antigen (HBsAg) test before recruitment. HBsAg-positive participants will not be recruited. For HBsAg-negative participants, the following algorithm applies: If the hepatitis B core antibody (HBcAb) is negative, the participant is eligible for recruitment. If HBcAb is positive, then the hepatitis B surface antibody (HBsAb) is tested. If both HBcAb and HBsAb are positive, the participant is eligible for recruitment. If HBcAb is positive but HBsAb is negative, the participant will not be recruited.

[0428] 7. Acute or chronic hepatitis C virus (HCV) infection (proven by antibody titer).

[0429] 8. Active systemic viral or fungal infection within 14 days prior to administration.

[0430] 9. A positive or indeterminate TB test indicates possible tuberculosis (TB) infection.

[0431] 10. Screening patients must have a history of latent or active TB within 8 weeks prior to their visit or have been exposed to endemic areas.

[0432] 11. Female subjects who are breastfeeding or sexually active and do not wish to use contraception and are not postmenopausal. Postmenopause is defined as amenorrhea for ≥12 consecutive months without other cause and with a recorded serum follicle-stimulating hormone level ≥40 mIU / mL and estradiol concentration ≤110 pmol / L in the 6 months prior to administration of the study drug.

[0433] 12. The serum pregnancy test is positive at the time of screening or on day -1.

[0434] 13. Serum creatinine is higher than the upper limit of normal (ULN) of the laboratory reference range at the time of screening or on day 1.

[0435] 14. At screening, alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > ULN of the laboratory reference range, or on day -1 > 1.5 × ULN of the laboratory reference range.

[0436] 15. Any of the following hematological results: at screening or day -1, hemoglobin <130 g / L for men, hemoglobin <115 g / L for women, hematocrit <0.37 L / L for men, hematocrit <0.33 L / L for women, white blood cell (WBC) count <3.0 × 10⁻⁶. 3 / μL, absolute neutrophil count <2.0×10 3 / μL, and platelet count <150 or >400×10 3 / μL. The clinical laboratory results of the complete blood count (CBC) on day-1 were considered clinically relevant and unacceptable by the researchers.

[0437] 16. During screening, a history of complement deficiency or complement activity below the normal reference range is evaluated using CAP ELISA.

[0438] 17. History of malignant disease, except for non-melanoma skin cancer or cervical carcinoma in situ that has been treated and shows no signs of recurrence.

[0439] 18. Participated in a clinical study within 30 days prior to the start of day 1 administration, or used any experimental small molecule therapy within 30 days prior to the start of day 1 administration.

[0440] 19. Participants must have completed more than one mAb clinical trial, or one mAb clinical trial within 12 months prior to screening, during which time the subject was exposed to the active investigational drug. Subjects who participated in only one mAb trial may be considered for recruitment if they completed the study more than 12 months prior to screening.

[0441] 20. Previously exposed to ALXN1210.

[0442] 21. Underwent major surgery or hospitalization within 90 days prior to administration.

[0443] 22. History of allergy to excipients of ALXN1210 (e.g., polysorbate 80).

[0444] 23. A documented history of penicillin or cephalosporin allergy.

[0445] 24. History of severe allergic reactions (allergic reactions or angioedema) to any product (food, medicine, etc.).

[0446] 25. Currently smokes more than 10 cigarettes per day (recruitment of former smokers may be permitted at the researcher's discretion).

[0447] 26. History of illicit drug abuse, a history of severe alcohol abuse within one year prior to the screening visit, or a history of frequent alcohol consumption (more than 14 units of alcohol per week [1 unit = 150 mL of wine, 360 mL of beer, or 45 mL of 40% alcohol]) within six months prior to the screening visit.

[0448] 27. The urine drug toxicology screening at the time of screening or on day -1 is positive.

[0449] 28. A positive breathalyzer test was obtained if alcohol was consumed within 48 hours prior to administration of the study drug or on day -1.

[0450] 29. Donated plasma within 7 days prior to administration. Donated or lost more than 50 mL of blood (excluding blood drawn during screening) within 30 days prior to administration, or donated or lost more than 499 mL of blood within 56 days prior to administration.

[0451] 30. History of continuous local, inhaled, or systemic use of steroids for >28 days within 90 days prior to drug administration, or history of any inhaled or local immunosuppressive therapy.

[0452] 31. Use of prescription drugs (excluding oral contraceptives) within 14 days prior to administration of the study drug, unless otherwise approved by the sponsor in advance.

[0453] 32. During the 14 days prior to administration of the study drug, regular use of over-the-counter medications, including herbal remedies and supplements, is permitted. Multivitamins, ≤2g of acetaminophen daily, and topical skin products with no significant systemic absorption are also permitted.

[0454] 33. Clinical diagnosis of any autoimmune or rheumatic disease (e.g., systemic lupus erythematosus, rheumatoid arthritis).

[0455] 34. During the study, immunization with a live attenuated vaccine should be administered 28 days prior to drug administration or planned vaccination (except for vaccinations planned in the study protocol). Immunization with inactivated or recombinant influenza vaccines is permitted.

[0456] 35. Fever (confirmed temperature > 37.6°C) occurring within 14 days prior to administration (e.g., fever associated with a symptomatic viral or bacterial infection).

[0457] 36. Subjects with any medical history, condition, or risk that the researchers believe could impede the subject's full participation in the study or adherence to the protocol, or could pose any other risk to the subject or confuse the assessment of the subject or the study results.

[0458] 6. Infect

[0459] To mitigate the risk of infection associated with terminal complement suppression, the following treatments were administered to the subjects in this study:

[0460] 1. MCV4 vaccination must be administered at least 56 days prior to ALXN1210 administration on day 1 (if the subject has not received MCV4 in the past 3 years, or if the subject has previously received it but there is insufficient documentation to confirm the previous vaccination).

[0461] 2. Administer two doses of serum-group B meningococcal vaccine. The first dose must be administered at least 56 days before Day 1 of the vaccine, and the booster dose must be administered at least 28 days before Day 1 of the vaccine. The interval between the first and second doses must be at least 28 days.

[0462] 3. Prophylactic antibiotic treatment: Oral penicillin V 500mg, twice daily (equivalent to 1×10⁻⁶ mg). 6 (units), until complement activity is normalized (determined by CH50 assay).

[0463] The first dose of antibiotic was administered orally on the night of Day 1 prior to the first day of the study (dose administration). For the outpatient portion of the study, subjects were instructed to take antibiotics approximately simultaneously (twice daily) on each scheduled date. Adherence to the antibiotic prophylaxis regimen was monitored daily using an appropriate system (such as sending text messages).

[0464] The following observations support the use of antibiotics for prophylaxis in this single-dose study:

[0465] 1. Penicillin is the first-line drug for eradicating Neisseria meningitidis from carriers.

[0466] 2. Complement deficiency patients who received monthly injections of benzathine penicillin G as a prophylaxis against recurrent meningococcal disease during a period of 2 to 4 years experienced significantly fewer episodes of Neisseria infection compared to deficiency individuals who did not receive prophylaxis (Figueroa JE et al., Clin. Microbiol. Rev. 1991 Jul; 4(3): 359-95).

[0467] 3. High levels of penicillin resistance caused by plasmid-encoded β-lactamases are rarely encountered in meningococcal strains (Yazdankhah SP et al., J. Med. Microbiol. Sep 2004; 53(Pt9): 821-32).

[0468] 4. Some physicians have already provided antibiotic prophylaxis with twice-daily oral administration of penicillin V 500 mg when treating patients with eculizumab for PNH and aHUS (Kelly RJ et al., Blood 2011 June 23; 117(25): 6786-92 and Leeds Teaching Hospitals NHS Trust, Kings College Hospital NHS Foundation Trust. National Specialised Commissioning Team (NSCT) Service Specification Paroxysmal Nocturnal Haemoglobinuria (PNH). 2013).

[0469] 5. Uncertainty about the effectiveness of vaccines in immunocompromised patients has prompted several countries (such as France) to recommend continuous antibiotic prophylaxis during eculizumab treatment in patients with PNH and aHUS (Zuber J, Fakhouri F, Roumenina LT, Loirat C, Fremeaux-Bacchi V. Use of eculizumab for atypical haemolytic uraemic syndrome and C3 glomerulopathies. Nat. Rev. Nephrol. 2012 Nov; 8(11): 643-57).

[0470] 7. Previous and accompanying medications and procedures

[0471] Record prior medications (any medications or substances taken by the subject within 14 days prior to signing the ICF until the administration of the study drug) and concomitant medications (any medications or substances taken by the subject after the administration of the study drug until the completion of the last study visit) on the subject's electronic case report form (eCRF). Record prior procedures (any therapeutic interventions [e.g., surgical / biopsy, physical therapy] performed within 14 days prior to signing the informed consent form until the administration of the study drug) and concomitant procedures (any therapeutic interventions [e.g., surgical / biopsy, physical therapy] performed after the administration of the study drug until the completion of the last study visit) on the subject's eCRF.

[0472] Concomitant therapy refers to any medication or substance administered from the start of the study screening until the completion of the last study visit. During the study, subjects should be instructed not to start taking any new medications, including over-the-counter drugs and herbal preparations, unless they obtain permission from the investigator. Occasional use of over-the-counter antipyretics or analgesics (e.g., acetaminophen) is permitted during the study.

[0473] Companion procedures are any therapeutic interventions (e.g., surgery / biopsy, physical therapy) or non-study diagnostic assessments (e.g., blood gas measurements, bacterial cultures) performed from the time the subject signs the informed consent form until the last study visit. Companion procedures are not permitted unless medically justified.

[0474] 8. Randomization and Blinding

[0475] Eligible subjects who meet the inclusion and exclusion criteria are assigned unique numbers for recruitment and randomization.

[0476] This is a partially blinded study, which makes:

[0477] • Group 1a. Dosage was double-blind (single 400 mg ALXN1210 SC or placebo SC). Subjects in group 1a were randomized in a 2:1 ratio (4 received ALXN1210 SC, 2 received placebo SC; N=6).

[0478] • Group 1b. The dosage was double-blind (a single 400 mg dose of ALXN1210 SC or placebo SC). Subjects in group 1b were randomized in a 5:1 ratio (20 received ALXN1210 SC and 4 received placebo SC; N=24).

[0479] • Group 2. Dosage (a single 400 mg dose of ALXN1210 IV) was open-label (N=12).

[0480] During group 2 administration, both the subjects and the on-site medical / nursing personnel were aware of the medication / dosage administered.

[0481] During dosing in groups 1a and 1b, subjects and on-site medical / nursing staff at the research center were unaware of the investigational drug allocation. Pharmacy staff preparing the SC injection were not unaware, nor were the personnel administering the investigational drug, while all other research center staff involved in the safety evaluation remained unaware of the investigational drug allocation. Sponsor personnel were not unaware as needed (e.g., to monitor the proper preparation of the SC injection, to determine the reportability of SAEs) and were prohibited from sharing any information regarding the investigational drug allocation with research center staff.

[0482] 9. Drug description for research

[0483] The research products are described in Table 16.

[0484] Table 16: Research Products

[0485]

[0486] 1 Each vial of ALXN1210 IV contains a nominal excess filler to ensure that 15 mL (150 mg of ALXN1210) can be dispensed for administration.

[0487] 2 Each vial of ALXN1210 SC pharmaceutical product includes a nominal excess filler to ensure that 1 mL (100 mg of ALXN1210) can be dispensed for administration.

[0488] Abbreviations: BP = British Pharmacopoeia; IV = Intravenous; NA = Not applicable; Ph Eur = European Pharmacopoeia; SC = Subcutaneous

[0489] 10. ALXN1210 and placebo

[0490] Each vial of ALXN1210SC contains 100 mg of ALXN1210 (100 mg / mL) in 50 mM sodium phosphate, 25 mM arginine, 5% sucrose, and 0.05% polysorbate 80. ALXN1210 SC is formulated to pH 7.4 and is provided as a fully prepared, sterile, preservative-free 100 mg / mL aqueous solution of ALXN1210, supplied in 2 mL disposable vials. Each vial of ALXN1210 SC includes a nominal excess filler to ensure that 1 mL (100 mg of ALXN1210) can be dispensed for administration.

[0491] Each dose of placebo SC contains 0.9% sodium chloride injection (Ph Eur or BP) in the same volume as specified in groups 1a and 1b.

[0492] Each vial of ALXN1210 IV contains 150 mg of ALXN1210 in 10 mM sodium phosphate, 150 mM sodium chloride, 0.02% polysorbate 80, and water for injection. ALXN1210 IV is formulated to pH 7.0 and is provided as a fully prepared, sterile, preservative-free aqueous solution of 10 mg / mL ALXN1210, supplied in 20 mL disposable vials. ALXN1210 IV is diluted in 0.9% sodium chloride injection (Ph Eur or BP) and administered via IV infusion at a maximum rate of 333 mL / hour, except for interruptions for safety or technical reasons.

[0493] Store ALXN1210 vials at 2°C to 8°C (36°F to 46°F) and away from light. Do not freeze or shake ALXN1210 vials.

[0494] ALXN1210 SC and placebo SC were prepared in a blinded manner in a syringe for SC administration. ALXN1210 SC or placebo SC was not diluted. ALXN1210 SC and placebo SC were placed directly into the syringe.

[0495] The ALXN1210 IV is designed for IV infusion at a maximum rate of 333 mL / h by dilution in commercially available saline (0.9% sodium chloride injection; Ph Eur or BP), except for interruptions for safety or technical reasons.

[0496] Dilute ALXN1210 IV with 0.9% sodium chloride injection (Ph Eur or BP) before administration. The shelf life of the dosing solution is 4 hours at room temperature (15°C to 25°C, 59°F to 77°F). The effective date and time of the dosing solution are calculated from the date the first vial is broken. Administer the dose within the effective date and time. Each 1-mL syringe of ALXN1210 SC or placebo SC (4 syringes per subject) should be drawn from the vial and administered within 1 hour.

[0497] 11. application

[0498] All doses of ALXN1210 SC or placebo SC were administered via four 1 mL injections of 100 mg SC into the abdominal region (Table 17). All four 1 mL injections were administered within 15 minutes, with at least 15 minutes between the end of one subject's injection and the start of the next.

[0499] Table 17: Dosage Reference Table for ALXN1210 SC and Placebo SC Formulations

[0500]

[0501]

[0502] All doses of ALXN1210 IV are administered via IV infusion at a maximum rate of 333 mL / h using an IV kit with tandem filters, except for interruptions for safety or technical reasons. There should be at least a 15-minute interval between the end of one subject's infusion and the start of the next subject's infusion.

[0503] Table 18: Dosage Reference Table for ALXN1210 IV Formulation

[0504]

[0505] 1 The infusion duration is an approximation.

[0506] 12. Management of potential adverse events during drug administration

[0507] Some subjects treated with intravenous monoclonal antibodies experienced infusion-related complications, the signs or symptoms of which could be classified as acute anaphylactic / hypersensitivity reactions or cytokine release syndrome.

[0508] During and after administration of the study drug, closely monitor subjects for any signs of allergy and other hypersensitivity reactions, including changes or cessation of circulation and / or respiration, or hives, arthralgia, myalgia, or other signs of related reactions. Appropriate treatment may be administered immediately. Infusion-related adverse events may occur, and depending on their type and severity, infusion may need to be interrupted. Inform subjects of early symptoms and signs of hypersensitivity reactions, including hives, swelling of the face, eyelids, lips, or tongue, or dyspnea. Use an acute infusion reaction algorithm to manage infusion-related reactions. In this study, periodic assessments will be conducted to monitor infusion reactions and infusion site reactions. To ensure timely management of reactions, at least 15 minutes will be allowed between the end of one subject's infusion / injection and the start of the next subject's infusion / injection. No more than 6 subjects will be administered daily. Treat any reactions and consider them in the dose continuation / escalation and toxicity rules. If an allergic reaction occurs, follow the current UK Treatment Guideline for Anaphylactic Reactions issued by the UK Resuscitation Council.

[0509] 13. Pharmacokinetic (PK) and pharmacodynamic (PD) assessment

[0510] Following drug administration, serum samples were collected at the following time points for determining serum ALXN1210 concentration, analyzing total and free C5 concentrations, cRBC hemolysis, and possibly other C5 activation measures. The actual serum sampling date and time were recorded and used for PK and PD calculations.

[0511] Serum concentrations of ALXN1210 were measured starting at the following sampling time points: before administration (within 15 minutes before the start of infusion / injection [SOI]); at the end of infusion / injection (EOI) on day 1, 30 minutes after EOI, and at the following time points after SOI: 2 hours, 4 hours, and 8 hours; day 2 (24 hours); day 3 (48 hours); day 5 (96 hours); day 8 (168 hours); day 15 (3... Day 36 (36 hours); Day 22 (504 hours); Day 29 (672 hours); Day 36 (840 hours); Day 43 (1008 hours); Day 50 (1176 hours); Day 57 (1344 hours); Day 71 (1680 hours); Day 90 (2136 hours); Day 120 (2856 hours); Day 150 (3576 hours); and Day 200 (4776 hours).

[0512] All subjects who provided a sufficient number of serum PK samples to characterize the concentration-time curve were included in the PK analysis population. All subjects who provided PD samples were included in the PD analysis population.

[0513] 14. Immunogenicity assessment

[0514] Serum samples were collected at the following time points: before administration (within 15 minutes before SOI), and on days 15 (336 hours), 29 (672 hours), 57 (1344 hours), 90 (2136 hours), 120 (2856 hours), 150 (3576 hours), and 200 (4776 hours), and ADA against ALXN1210 was analyzed. Further characterization of the antibody response was performed, as needed, based on PK / PD and safety data for ALXN1210.

[0515] All subjects who provided pre- and post-dose samples of ADA were included in the immunogenicity analysis population.

[0516] Immunogenicity assays evaluated the anti-drug antibody (ADA) against ALXN1210. Detailed procedures for collecting, processing, storing, and transporting serum samples for immunogenicity analysis are provided in the laboratory manual.

[0517] 15. Security assessment

[0518] Safety assessments included TB testing, physical examination results, vital sign measurements, immunogenicity (ADA) testing, laboratory evaluations, ECG, infusion and injection site evaluations (e.g., bleeding, bruising, erythema, swelling, induration, and pain), and adverse event monitoring. Adverse events were graded according to the National Cancer Institute's Common Terminology for Adverse Events v4.03 (CTCAE v4.03), published on June 14, 2010. Laboratory evaluations included hematology, chemistry, and coagulation panels; classified CBC; urinalysis; and serum pregnancy testing in female subjects.

[0519] Perform clinical and laboratory evaluations to assess the safety of ALXN1210. The evaluation schedule is described in the evaluation timeline. Track any abnormal results until they subside or stabilize.

[0520] As described in the assessment form, review demographic parameters, including age, sex, race, and ethnicity. Obtain a complete medical history.

[0521] Vital signs, including temperature (°C; oral temperature), respiratory rate, supine blood pressure, and pulse, were measured after the subjects rested in a supine or semi-recumbent position for at least 5 minutes. The timing of vital sign measurements was described in the assessment schedule. Blood pressure or pulse measurements outside the range were repeated at the investigator's discretion. Any confirmed, clinically significant vital sign measurements were recorded as adverse events.

[0522] Record weight, height, and BMI as described in the assessment schedule. Each examination includes the following assessments: general appearance; skin; head, ears, eyes, nose, and throat; neck; lymph nodes; chest; heart; abdomen; limbs; central nervous system; and musculoskeletal system.

[0523] Three 12-lead ECGs were obtained after subjects rested for at least 5 minutes. The timing of the ECGs is described in the assessment schedule. Additionally, continuous cardiac recordings were performed at each dose administration, from pre-dose to the end of the IV infusion in group 2a, and from pre-dose to 3 hours after the end of the SC injection in groups 1a and 1b. Heart rate, PR, QRS, RR, and QT were measured, and the corrected QTcF interval was calculated.

[0524] As described in the assessment schedule, blood samples were collected for hematological, clinical chemistry, coagulation, and virological serological analyses, as well as urine samples for urinalysis, urine chemistry analysis, and drug and alcohol screening.

[0525] The following hematological parameters of the blood sample were analyzed: platelet count, red blood cell (RBC) count, and white blood cell (WBC) count; automatic classification (neutrophils, lymphocytes, monocytes, eosinophils, basophils); hemoglobin; hematocrit; and RBC indices (mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration). The timing of the hematological assessments is described in the assessment schedule.

[0526] The following clinical chemistry parameters were analyzed in blood samples: blood urea nitrogen; creatinine; glucose; sodium; phosphorus; potassium; chloride; total carbon dioxide; total calcium; magnesium; AST; ALT; gamma-glutamyl transferase; alkaline phosphatase; lactate dehydrogenase; total bilirubin, direct and indirect bilirubin; uric acid; albumin; and total protein. Indirect bilirubin is calculated based on total bilirubin and direct bilirubin values; if direct bilirubin is below the quantification limit, indirect bilirubin results cannot be obtained.

[0527] Serum follicle-stimulating hormone (FSH) levels and estradiol concentrations were measured during screening of postmenopausal female subjects to confirm their postmenopausal status.

[0528] The assessment timeline describes the schedule for the chemical assessment.

[0529] Prothrombin time, international normalized ratio, and partial thromboplastin time were analyzed in blood samples. The timing of coagulation assessments is described in the assessment schedule.

[0530] Urinalysis included specific gravity, pH, glucose, protein, blood, and ketones. Microscopic examination of urine samples was performed only for abnormal results. Urine samples were also sent to a pathology laboratory to measure protein and creatinine to calculate the urinary protein:creatinine ratio. The timing of urinalysis and urinary chemistry assessments is described in the evaluation schedule.

[0531] Analyze the HIV-1, HIV-2, HBsAg, and HCV antibody titers in blood samples collected during screening. All participants must undergo a hepatitis B surface antigen test before recruitment. HBsAg-positive participants are not recruited. For HBsAg-negative participants, the following testing algorithm is required:

[0532] 1. If HBcAb is negative, the subject meets the recruitment criteria.

[0533] 2. If HBcAb is positive, then test for hepatitis B surface antibody (HBsAb).

[0534] a. If both HBcAb and HBsAb are positive, the subject meets the recruitment criteria.

[0535] b. If HBcAb is positive and HBsAb is negative, the subject will not be recruited.

[0536] The following compounds were analyzed in urine samples used for drug screening: amphetamines, barbiturates, benzodiazepines, cocaine, methadone, opioids, phencyclidine, methamphetamine, 3,4-methylenedioxy-deoxyephedrine, and tetrahydrocannabinol (cannabinoids). An alcohol breath test was performed. If the result was positive before administration, no medication was administered. The timing of urine drug testing and the alcohol breath test is described in the evaluation schedule.

[0537] Pregnancy testing (β-human chorionic gonadotropin) was performed in all female subjects. The timing of the pregnancy testing is described in the evaluation schedule.

[0538] As described in the evaluation schedule, serum samples were collected for the QuantiFERON-TB test.

[0539] Complement activity was measured during screening, including appropriate assays such as CAP ELISA / C5 (hemolysis) inhibition, to confirm that subjects did not have complement deficiency. Subjects found to have complement deficiency were excluded from the study.

[0540] Serum samples were collected at baseline and during follow-up to measure CH50 activity using in vitro LIA to confirm the normalization of complement activity. If a normal CH50 result was obtained from the first CH50 sample collected from the subject during follow-up, antibiotic prophylaxis could be discontinued, and a second, pre-planned CH50 sample was not required. If the first and second CH50 samples were abnormal, the baseline sample could be analyzed, and additional CH50 samples could be obtained until complement activity was restored.

[0541] Serum bactericidal antibody (SBA) titers against serogroups A, C, W135, and Y meningococci were obtained during screening. Titer measurements were used to exclude subjects without an immune response from receiving the drug.

[0542] Evaluate the subcutaneous injection or IV infusion site. Assess pain at the SC or IV infusion site using the Visual Analogue Scale (0-10). No pain assessment was performed prior to administration. Induration or reactions of 1 cm in size are not considered adverse events unless they persist for more than 24 hours.

[0543] Analyze anti-drug antibodies (ADAs) in serum samples. The assessment timeline describes the schedule for ADA serum sample collection.

[0544] 16. Adverse event management

[0545] Researchers are responsible for detecting, evaluating, recording, and reporting all adverse events (AEs). All AEs must be recorded from the time informed consent is signed until the study is completed. There is no time limit for AEs deemed causally related.

[0546] Regardless of causality, all observed or voluntarily provided adverse events (AEs) are reported and recorded in the data capture system. Adverse events reported by subjects and / or their parents or legal guardians, and / or identified based on open-ended questions posed by the researchers, or revealed through observation, physical examination, or other research procedures are collected and recorded.

[0547] An adverse event (AE) is defined as any adverse and unexpected sign (e.g., including abnormal laboratory results), symptom, or illness that occurs during a clinical study and is temporarily related to the use of a pharmaceutical product or procedure, regardless of whether it is believed to be related to the pharmaceutical product or procedure.

[0548] Any exacerbation of a chronic or intermittent pre-existing condition, including an increase in the frequency and / or intensity of that condition, is considered an adverse event (AE).

[0549] Abnormal test results are considered adverse events (AEs). If an abnormal laboratory value is identified, researchers are strongly advised to report the diagnosis, sign, or symptom, rather than the abnormal test value alone. An abnormal test result is recorded as an AE if any of the following conditions are met: it is related to a sign or symptom; it requires an additional diagnostic test (repeat tests are not considered additional tests); it requires medical or surgical intervention; it results in a change in study dosing beyond the protocol-defined dosing range or causes study interruption; it requires substantial additional treatment; or it does not meet any of the above conditions.

[0550] The definition also includes signs or symptoms caused by the following reasons: drug overdose, drug withdrawal, drug misuse, drug interactions, extravasation, exposure during pregnancy, exposure through breastfeeding, drug misadministration, and occupational exposure.

[0551] AE does not necessarily include the following:

[0552] • Medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, intravenous infusion); conditions leading to the procedure are AEs (e.g., laparoscopic cholecystectomy is a procedure or treatment for necrotizing gallbladder SAE).

[0553] • Pre-existing diseases or symptoms that are present at the time of screening evaluation or detected before the screening evaluation and have not worsened.

[0554] • Situations where no unfortunate medical incidents have occurred (e.g., hospitalization for elective surgery if planned before the study begins; social and / or convenient admission).

[0555] Any AE that meets any one of the following criteria should be recorded as an SAE.

[0556] SAE is defined as any unfortunate medical incident that produces the following results at any dose:

[0557] 1. Causes death

[0558] 2. Life-threatening a

[0559] 3. Requires hospitalization or extended hospitalization. b Hospitalization does not necessarily include the following:

[0560] • Rehabilitation / Hospice / Nursing Facilities

[0561] Emergency room visits within 24 hours

[0562] • Elective or pre-planned admission / surgery / same-day surgery

[0563] • The hospital admission plan is specified

[0564] • Admitted due to pre-existing symptoms not related to new adverse events (AEs) or worsening of existing AEs.

[0565] 4. Leading to persistent or significant disability / loss of ability

[0566] 5. Congenital abnormalities / birth defects

[0567] 6. Important medical events c

[0568] a In the definition of “serious,” the term “life-threatening” refers to an event in which the subject is at risk of death when the event occurs; it does not refer to an event that, hypothetically, could lead to death if it were more serious.

[0569] b Hospitalization requires hospitalization or extension of existing hospitalization. AEs (Adverse Events) associated with hospitalization or extension of hospitalization are considered SAEs.

[0570] cSignificant medical events: In determining whether expedited reporting is appropriate in other circumstances, medical and scientific judgment should be exercised. These other circumstances may not be immediately life-threatening or result in death or hospitalization, but may endanger the subject or may require intervention to prevent one of the other consequences listed in the above definition. These should generally also be considered serious. Examples of such events include intensive treatment of anaphylactic bronchospasm in the emergency room or at home; blood dysregulation or seizures that do not result in hospitalization; or the development of drug dependence or drug abuse.

[0571] Severity and severity should be distinguished. Severity describes the intensity of an AE, while the term severity refers to an AE that has met the SAE criteria as described above.

[0572] All AEs are classified according to the following criteria from CTCAE v4.03, which was released on June 14, 2010.

[0573] • Grade 1: Mild (aware of signs or symptoms, but easily tolerable)

[0574] Level 2: Moderate (discomfort sufficient to interfere with normal activities)

[0575] Level 3: Severe (loss of ability, unable to perform normal activities)

[0576] Level 4: Life-threatening

[0577] Level 5: Lethal

[0578] Record changes in the severity of adverse events (AEs) to assess the duration of AEs at each intensity level being evaluated. If the severity of intermittent events varies, adverse events characterized as intermittent require recording the onset and duration of each episode.

[0579] A causality assessment by researchers was provided for all adverse events (both non-severity and severity). This assessment was recorded in the data capture system and, where appropriate, on any other forms. The definition of causality assessment is as follows:

[0580] • Irrelevant (unrelated): This relationship indicates that there is no connection between the research product and the reported event.

[0581] • Unlikely to be related: This relationship indicates that the clinical presentation is highly consistent with causes other than the study product, but cannot be attributed with absolute certainty, and the relationship between the study product and the AE cannot be completely ruled out.

[0582] • Possibly related: This relationship suggests that treatment with the investigational product may have caused or contributed to an adverse event (AE); that is, the event follows a reasonable chronological order from the time of administration of the investigational drug, and / or follows a known response pattern to the investigational product, but may also be caused by other factors.

[0583] • Highly likely related: This relationship indicates a plausible chronological sequence between the event and the administration of the study product, and a possible association between the event and the study product. This will be based on the known pharmacological effects of the study product, known or previously reported adverse reactions to the study product or drug class, or the judgment of the researcher's clinical experience.

[0584] • Clear Relevance: The event is related to the time frame of the research product. Other circumstances (comorbidities, adverse drug reactions, or progression / manifestation of disease states) do not appear to explain this event; the event corresponds to known drug properties; improvement upon interruption; and recurrence upon reactivation.

[0585] If a subject experiences a SAE that results in death, the following procedure is followed: the SAE leading to death is recorded as a death / fatal consequence, with the end date being the date of death. If the subject had other persistent AEs / SAEs at the time of death, these events are recorded as persistent, without an end date. Unless otherwise stated in the autopsy report or by the researchers, only one event has a death / fatal consequence.

[0586] 17. statistics

[0587] Before locking down the database, develop and finalize the formal statistical analysis plan (SAP).

[0588] The safety cohort consisted of all subjects who received at least one dose of the study drug. Subjects in this cohort were used for safety analysis.

[0589] The PK population consists of all subjects with sufficient serum concentration data to allow for the calculation of PK parameters. The PK population is used for PK pooling.

[0590] The PD cohort consisted of all subjects with sufficient total C5 and free C5 concentration data, as well as cRBC hemolysis data. The PD cohort was used for PD pooling.

[0591] The immunogenicity analysis population consisted of all subjects for whom ADA samples were collected before and after administration.

[0592] A total evaluable sample size of 36 subjects (24 ALXN1210 SC subjects from cohort 1 and 12 ALXN1210 IV subjects from cohort 2) provided >80% power to extrapolate a hypothetical absolute bioavailability of 0.6 with a coefficient of variation of 0.35, and a lower limit of the 90% confidence interval for the bioavailability ratio of ALXN1210 SC to IV >0.4. Additionally, 6 subjects received placebo SC (2 from cohort 1 and 4 from cohort 1b). Cohort 1a was randomized in a 2:1 ratio to receive either ALXN1210 SC or placebo SC, and cohort 1b was randomized in a 5:1 ratio. This brought the total planned number of subjects to N = 42.

[0593] Generally, descriptive statistics for continuous variables include the number of non-missing values, arithmetic mean, standard deviation, median, minimum, and maximum. Descriptive statistics for PK parameters include the number of observations, arithmetic mean, standard deviation, arithmetic coefficient of variation (%CV), median, minimum, maximum, geometric mean, and geometric %CV. Categorical variables are summarized using percentage and frequency counts by group and time point.

[0594] All participants were included in the participant treatment summary, which summarized the frequency and percentage of participants who completed or discontinued the study by cohort, along with the reasons for discontinuation. Demographic information and baseline characteristics of all participants were summarized by cohort and population.

[0595] Safety analyses were performed on the safety cohorts and reported for each cohort. Safety analyses included analyses of all AEs, ECGs, clinical laboratory data, physical examinations, and vital sign measurements, presented using descriptive statistics. No inferential statistical analyses were planned for the safety parameters of this study. The incidence of AEs and SAEs occurring during treatment was summarized by system organ classification and priority, and by relationship to the study drug, for each cohort and overall study. AEs occurring during treatment were also summarized by severity, by cohort and overall study. Serious AEs and AEs leading to study withdrawal were listed. Subjects with multiple AEs in a category (e.g., overall, system organ classification, priority) were counted once within that category. For the severity table, the most severe event in a subject's category was counted.

[0596] Changes in vital signs measurements and laboratory assessments (e.g., chemical, differential CBC, and urinalysis) relative to baseline were summarized for each group. Laboratory parameter values ​​were graded according to CTCAE classification. Transition tables were generated for these laboratory parameters by group. These tables summarized the number of subjects with each baseline grade relative to a reference range, as well as the change to the worst-to-highest grade assessed post-dose during the study period.

[0597] ECG parameters, including heart rate, PR, RR, QRS, QT, and corrected QTcF interval, were measured at specified time points. The mean of the three ECG readings collected at each time point was calculated, and the change relative to the pre-treatment baseline was assessed for each cohort.

[0598] Outlier analysis was performed, and the frequency and percentage of subjects who met any of the following outlier criteria at each visit were summarized by group:

[0599] • QT, QTcF interval > 450 milliseconds

[0600] • QT, QTcF interval > 480 milliseconds

[0601] • QT, QTcF interval > 500 milliseconds

[0602] • The QT and QTcF intervals increase by >30 milliseconds relative to the baseline.

[0603] • The QT and QTcF intervals increase by >60 milliseconds relative to the baseline.

[0604] All accompanying medications were coded using the WHO Drug Dictionary, which summarizes the frequency and percentage of accompanying medications.

[0605] Using Phoenix WinNonlin 6.3 or later, individual serum concentration data (including actual sampling date and time) of subjects treated with ALXN1210 were used to derive PK parameters via a non-compartmental analysis method.

[0606] The following PK parameters were obtained: maximum observed serum concentration (C max The time to reach the maximum observed serum concentration (T) max The area under the serum concentration-time curve (AUC) from zero to the final quantifiable concentration. t The area under the curve from time zero to infinity (AUC) ∞ ), terminal elimination rate constant (λ) z Terminal elimination half-life (T) 1 / 2 Total clearance (CL or CL / F) and distribution volume (V) d or V d / F).

[0607] For C max AUC t and AUC ∞The geometric mean ratio (ALXN1210 SC / ALXN1210 IV) and its 90% CI were calculated and tabulated. CI was calculated using between-subjects variance. Concentration assessments over time are presented.

[0608] The PD efficacy of ALXN1210 SC and IV was evaluated by assessing other measures of serum total and free C5 concentrations, cRBC hemolysis, and C5 activation over time. Collected samples were analyzed as described in the evaluation schedule.

[0609] Immunogenicity as measured by ADA is summarized in tabular form by group and subject list.

[0610] Example 3: Results of a Phase 1 study evaluating subcutaneous administration versus intravenous administration of a single dose of ALXN1210 in healthy subjects.

[0611] The following is a summary of data from a single-dose Phase 1 study conducted essentially as described in Example 2. Specifically, this study was designed to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of a single subcutaneous dose of 400-mg ALXN1210 compared to a single intravenous dose of 400-mg ALXN1210 or a subcutaneous placebo in 42 healthy subjects.

[0612] 1. Treatment of subjects

[0613] Of the 161 participants screened, 42 (26.09%) were randomly assigned to receive the investigational drug: placebo SC (n=6), ALXN1210 SC (n=24), and ALXN1210 IV (n=12). Figure 48 None of the randomly assigned participants prematurely ended the study.

[0614] 2. Plan Deviation

[0615] Of the 36 participants, at least one protocol deviation was reported (placebo SC: n=6; ALXN1210 SC: n=20; and ALXN1210 IV: n=10). Categories of protocol deviations included time window bias, participant compliance, failure to assess, exclusion criteria, and dosage administration.

[0616] In two subjects in the ALXN1210 IV group, protocol deviations were assessed as significant. In one subject, day 29 ADA, PK, PD, and laboratory assessments were not performed because the subject did not attend follow-up appointments. In the other subject, day 71 PK and PD samples were not collected because the subject did not attend follow-up appointments. Although these deviations were assessed as significant due to the nature of the study design (PK-related primary endpoint), they were not considered to have affected the interpretation of the results. Other protocol deviations were not considered to have affected the interpretation of the results or the safety of the subjects. Serum pregnancy test results were negative in all subjects during the study.

[0617] 3. Pharmacokinetic, pharmacodynamic and immunogenicity evaluation

[0618] All 42 randomly assigned subjects received the study drug and were included in the safety cohort (Table 19). By definition, all of these subjects were also included in the PD cohort and the immunogenicity analysis cohort. The 36 subjects in the safety cohort who received ALXN1210 SC or ALXN1210 IV had sufficient serum concentration data to calculate PK parameters and were included in the PK cohort (Table 19).

[0619] Table 19: Analysis Population (All Randomized Subjects)

[0620]

[0621] Note: Percentage (%) equals n / N × 100.

[0622] Abbreviations: IV = intravenous; N = total number of subjects; n = number of subjects; SC = subcutaneous.

[0623] 4. Demographics and other baseline characteristics

[0624] In all treatment groups, the majority of participants were male (66.7%) and white (69.0%), with a mean (±SD) age of 35.0 (±7.65) years. The mean (±SD) BMI for the overall population was 24.035 (±3.1582). Generally, the demographics were balanced across the treatment groups (Table 20).

[0625] Table 20: Demographic Statistics - Descriptive Statistics by Treatment (Safety Group)

[0626]

[0627]

[0628] Note: Percentage (%) equals n / N × 100.

[0629] Abbreviations: BMI = Body Mass Index; IV = Venous; max = Maximum value; min = Minimum value; N = Total number of subjects; n = Number of subjects; SC = Subcutaneous; SD = Standard deviation.

[0630] In the ALXN1210 SC group, 5 (20.8%) subjects reported using prior medications. No prior medication use was reported in the placebo SC group or the ALXN1210 IV group. 3 (50.0%), 13 (54.2%), and 8 (66.7%) subjects in the placebo SC group, ALXN1210 SC group, and ALXN1210 IV group, respectively, reported using concomitant medications. The most commonly used concomitant medications were aniline derivatives used to treat adverse events, such as acetaminophen / paracetamol (15 subjects), followed by progestins and estrogens, a fixed combination used for contraception (7 subjects). It is expected that reported concomitant medications will not affect the study results.

[0631] No subjects received any non-pharmacological therapies or procedures. All doses of ALXN1210 SC or placebo SC were administered via four 100-mg SC injections (1 mL each) over the abdominal area. All doses of ALXN1210 IV were administered via IV infusion using an IV kit with a tandem filter. All subjects received their assigned dose.

[0632] 5. Tabulation of pharmacokinetic, pharmacodynamic, and immunogenicity results and individual subject data

[0633] PK analysis was performed on the PK group, which consisted of all subjects from the safety group who received ALXN1210 SC or ALXN1210 IV and had sufficient serum concentration data to calculate PK parameters.

[0634] Figures 49-50 The mean (±SD) serum concentration-time curves (linear and log-linear scales) of healthy subjects after SCX and IV administration of ALXN1210 were presented. Linear scales were used separately. Figure 49 ) and log-linear scale ( Figure 50 The graph shows the relationship between the serum concentrations of ALXN1210 and the nominal time.

[0635] Table 21 summarizes the pharmacokinetic parameters of ALXN1210 after SC and IV administration. A total of 24 subjects received ALXN1210 SC administration; the median (range) t after SC injection... max The duration was 169.8 (96.0 to 508.1) hours. The geometric mean (CV%) t was for ALXN1210 SC and IV applications, respectively. 1 / 2Similarities were observed at 31.3 (13.6) days and 29.9 (15.4) days. Elimination of ALXN1210 was similar between the IV and SC pathways. Figure 49 ).

[0636] Table 21: Summary of Pharmacokinetic Parameters of ALXN1210 (Pharmacokinetic Group)

[0637]

[0638] a Presented t max of the median.

[0639] Note: For ALXN1210 SC treatment, CL and V d The columns represent CL / F and V respectively. d / F.

[0640] Abbreviation: AUC t =Area under the serum concentration-time curve from 0 to the final quantifiable concentration; AUC ∞ =Area under the serum concentration-time curve extrapolated from 0 to infinity; CL or CL / F = Systemic clearance of the drug from serum; C max = Maximum observed serum concentration; CV = Coefficient of variation; h = Hour; IV = Intravenous; L = Liter; max = Maximum value; min = Minimum value; n = Number of subjects; NA = Not applicable; SC = Subcutaneous; t 1 / 2 =Terminal elimination half-life; t max =Time to reach maximum observed serum concentration; V d or V d / F = Distribution volume; λ z =Terminal elimination rate constant.

[0641] Table 22 summarizes the absolute bioavailability of ALXN1210 SC. After logarithmic transformation of the data, statistical analysis was performed using a mixture model to determine the PK parameter (Cp) of ALXN1210 SC. max AUCt and AUC ∞ Compare with the reference (ALXN1210 IV). The C of ALXN1210 (SC / IV) is... max The GMR was 26.1% (95% CI: 21.3, 32.0). The absolute bioavailability of ALXN1210 SC, determined by GMR (SC / IV) based on AUC∞ estimates, was 60.4% (95% CI: 49.7, 73.3).

[0642] Table 22: Statistical Analysis of Absolute Bioavailability of ALXN1210

[0643] Subcutaneous (pharmacokinetic)

[0644]

[0645]

[0646] Note: The ratio is defined as the geometric mean of the ALXN1210 SC group divided by the geometric mean of the ALXN1210 IV group × 100. A linear mixture model with subject fixed and random effects was used.

[0647] Abbreviation: AUC t =Area under the serum concentration-time curve from 0 to the final quantifiable concentration; AUC ∞ =Area under the serum concentration-time curve extrapolated from 0 to infinity; CI = Confidence interval; C max = Maximum observed serum concentration; GMR = Geometric mean ratio; h = Hour; IV = Intravenous; n = Number of subjects; SC = Subcutaneous.

[0648] PD analysis was performed on the PD group, which consisted of all subjects from the safety group, who had sufficient data on free and total C5 concentrations and cRBC hemolysis.

[0649] Figure 51 The mean (±SD) percentage change in serum free C5 concentration over time relative to baseline was depicted in subjects receiving placebo SC, ALXN1210 SC, and ALXN1210 IV. After placebo administration of SC, mean free C5 remained relatively constant. A single dose of ALXN1210 IV 400 mg caused immediate and almost complete inhibition of free C5 (≥99%) until day 8 post-IV administration. A single dose of ALXN1210SC 400 mg also caused a reduction in free C5, but not to the same extent as seen after IV administration or with a less rapid onset. The maximum mean inhibition of free C5 (77%) was observed 1 week post-administration of ALXN1210SC.

[0650] The duration and extent of the decrease in average free C5 concentration depend on the exposure. Figure 52 The mean (±SD) percentage change in serum total C5 concentration over time relative to baseline was depicted in subjects receiving placebo (SC), ALXN1210 SC, and ALXN1210 IV. After placebo administration, the mean total C5 concentration remained relatively constant. However, administration of a single dose of ALXN1210 400 mg resulted in a maximum mean increase in total C5 relative to baseline of 82% and 107%, respectively, after SC and IV administration.

[0651] Figure 53This study depicts the change over time in the mean (±SD) percentage of chicken red blood cell (cRBC) hemolysis relative to baseline in subjects administered placebo SC, ALXN1210 SC, and ALXN1210 IV. Mean cRBC hemolysis remained relatively constant after placebo administration of SC. A single dose of ALXN1210 IV 400 mg caused immediate inhibition of mean cRBC hemolysis, with a maximum mean reduction of 88%. A single dose of ALXN1210 SC 400 mg also caused inhibition of cRBC hemolysis, but not to the same extent or with a less rapid onset compared to IV administration. A maximum mean inhibition of 29% of cRBC hemolysis was observed approximately 1 week after ALXN1210 SC administration. The duration and extent of cRBC inhibition depended on the exposure.

[0652] Immunogenicity analysis was performed on the immunogenicity assay group, which consisted of all subjects from the safety group whose ADA samples were collected before and after administration. Anti-antibody assays were performed before administration and at days 15, 29, 57, 90, 120, 150, and 200 after administration.

[0653] One subject in the ALXN1210 SC treatment group (subject 0344-185) had a confirmed ADA-positive sample at baseline (before administration) and all post-administration samples were also ADA-positive. All post-administration antibody titers for this subject were lower than the pre-administration titers. In this subject, the positive anti-antibody response was not considered clinically significant or ALXN1210-related. Therefore, this subject was not included in the immunogenicity summary provided below.

[0654] Four subjects (3 / 23 [13%] in the ALXN1210 SC group and 1 / 12 [8.3%] in the ALXN1210 IV group) developed ADA during treatment. In the ALXN1210 SC group: the first subject was ADA-positive at days 57, 90, 120, 150, and 200. Eculizumab cross-reactivity was positive for all ADA-positive values. The second subject was ADA-positive at days 29, 57, 90, 120, 150, and 200. Eculizumab cross-reactivity was positive for all ADA-positive values ​​except for day 90. The third subject was ADA-positive at days 90, 120, 150, and 200. Eculizumab cross-reactivity was positive for all ADA-positive values.

[0655] In the ALXN1210 IV group: one subject was ADA-positive at days 15, 29, 90, 120, 150, and 200. All ADA-positive subjects were eculizumab cross-reactive.

[0656] For SC and IV administration, the earliest positive ADA responses were observed on day 29 and day 15, respectively. ADA titers in ADA-positive samples were low, ranging from <1.0 to 27. ADA cross-reacted with eculizumab in most ADA-positive samples after SC administration. ADA did not cross-react with eculizumab after IV administration. All ADA-positive subjects remained positive until the end of the follow-up period. Due to the small number of ADA-positive subjects, the effect of ADA on PK and PD could not be formally assessed. Limited individual PK and PD results in these subjects indicated that immunogenicity had no significant effect on the PK or PD of ALXN1210.

[0657] 6. Pharmacokinetic, pharmacodynamic, and immunogenicity conclusions

[0658] Median (range) t after SC injection max The effective duration was 169.8 (96.0 to 508.1 hours). Following ALXN1210 SC and IV administration, the geometric mean terminal elimination half-life was similar at 31.3 days and 29.9 days, respectively.

[0659] C max The estimated GMR (SC / IV) was 26.1% (95% CI: 21.3, 32.0). Based on AUC, the estimated GMR (SC / IV) of ALXN1210 SC was 60.4% (95% CI: 49.7, 73.3).

[0660] The extent and duration of PD response, assessed by serum concentrations of free and total C5 and cRBC hemolysis, depended on exposure. A single dose of ALXN1210 IV 400 mg caused immediate and almost complete inhibition of free C5 (≥99%) until day 8 post-administration. A single dose of ALXN1210 SC 400 mg (administered as four 100 mg SC injections) also caused a reduction in free C5, but not to the same extent as IV administration or with a less rapid onset. The maximum mean inhibition of free C5 was 77%, occurring approximately one week after SC administration. Administration of 400 mg ALXN1210 resulted in a maximum mean increase of 82% and 107% relative to baseline in total C5, respectively, following SC and IV administration. A single dose of ALXN1210 IV 400 mg caused immediate inhibition of mean cRBC hemolysis, with a maximum mean reduction of 87%. Administration of a single dose of ALXN1210 SC 400mg also induced inhibition of cRBC hemolysis, but not to the same extent or with a less rapid onset compared to IV administration. A maximum mean inhibition of cRBC hemolysis of 29% was observed approximately 8 days after SC administration.

[0661] In the ALXN1210 SC and ALXN1210 IV groups, 3 / 23 (13%) and 1 / 12 (8.3%) subjects, respectively, reported ADA during treatment, with low ADA titers ranging from <1.0 to 27. The earliest post-dose ADA response was observed on day 29 and day 15, respectively, for SC and IV administrations. Following SC administration, ADA cross-reacted with eculizumab in most ADA-positive samples. Following IV administration, ADA did not cross-react with eculizumab. All ADA-positive subjects remained positive until the end of the follow-up period. Immunogenicity had no significant effect on the PK or PD of ALXN1210.

[0662] Another subject in the ALXN1210 SC treatment group had a confirmed ADA-positive sample at baseline (before administration) and all post-administration samples were also ADA-positive. All post-administration antibody titers in this subject were lower than the pre-administration titers. In this subject, the positive anti-antibody reaction was not related to ALXN1210.

[0663] 7. Exposure level

[0664] All subjects receiving a single dose of the study drug were included in the safety group (N=42): placebo SC (n=6); ALXN1210 SC (n=24); and ALXN1210 IV (n=12). In each subject assigned to receive ALXN1210 IV, a total infusion volume (80 mL) of the study drug was administered. In one subject, the infusion was interrupted for one minute because the programmed time to the pump was insufficient for complete infusion. In each subject receiving either ALXN1210 SC or placebo SC, a total volume of the study drug (4 mL) was administered.

[0665] 8. Adverse events

[0666] In all three treatment groups, 35 / 42 (83.3%) of subjects experienced 75 TEAEs (all Grade 1). The proportions of subjects with at least one TEAE were 91.7%, 83.3%, and 79.2% in the ALXN1210 IV, placebo SC, and ALXN1210 SC groups, respectively. No deaths or SAEs were reported during the study. No subjects discontinued the study drug or withdrew from the study due to TEAEs (Table 23). 。 During the study, all TEAEs subsided. Most TEAEs did not require any medication, and no non-pharmacological intervention was needed at any time.

[0667] Table 22: Treatment-Acting Adverse Events (TEAEs) - Overall Summary (Safety Group)

[0668]

[0669] Note: Percentage (%) equals n / N × 100.

[0670] Level 1 = Mild; Level 2 = Moderate; Level 3 = Severe; Level 4 = Life-threatening or disabling; Level 5 = Death related to TEAE.

[0671] Relevant TEAEs = TEAEs that are likely to be relevant, very likely to be relevant, or clearly relevant; Irrelevant TEAEs = TEAEs that are irrelevant or unlikely to be relevant.

[0672] a For ALXN1210 IV, a TEAE is considered to have occurred during the administration of the study drug if it occurs during infusion; for placebo SC and ALXN1210 SC, an adverse event is considered to have occurred during the administration of the study drug if it occurs between the first and last injection.

[0673] Abbreviations: E = number of events; IV = intravenous; N = total number of subjects at risk; n = number of subjects with adverse events (AEs); SC = subcutaneous; SAE = serious adverse event; TEAE = adverse event occurring during treatment.

[0674] A total of 75 treatment-related adverse events (TEAEs) were reported by 35 participants. The most frequently reported TEAEs across all treatment groups were nasopharyngitis (23 / 42 participants, 54.8%) and headache (7 / 42 participants, 16.7%). All TEAEs are summarized in Table 23 by system organ classification (SOC) and treatment priority.

[0675] Table 23: Frequency of Adverse Events During Treatment - Listed by System Organ Classification and Priority (Safety Group)

[0676]

[0677]

[0678] Note: Percentage (%) equals n / N × 100.

[0679] For a given SOC and "priority", each subject is counted only once, regardless of the actual number of adverse events that occur.

[0680] The classification of SOC and "priorities" is based on MedDRA v20.0.

[0681] Abbreviations: E = Events; IV = Intravenous; MedDRA = Dictionary of Medical Regulatory Activities; N = Total number of subjects at risk; n = Number of subjects with adverse events; SC = Subcutaneous; SOC = System Organ Classification; TEAE = Treatment-related adverse events.

[0682] Most TEAEs (72 / 75 TEAEs, 96%) were considered unrelated to ALXN1210 treatment. In the treatment group, 3 / 42 (7.1%) subjects reported 3 TEAEs, which were assessed by the researchers as ALXN1210 treatment-related (“potentially related”) and graded as Grade 1 (mild): (1) an upper respiratory tract infection in one subject from the ALXN1210 SC group, (2) a migraine in one subject from the ALXN1210 SC group, and (3) a headache in one subject from the ALXN1210 IV group. All 75 TEAEs were classified as Grade 1 (mild). No subjects died, experienced SAEs, or discontinued study medication or the study due to TEAEs.

[0683] Generally, the mean values ​​of hematology, coagulation, blood chemistry, urinalysis, and urine chemistry analysis are within the reference range, and there is no obvious trend in the mean values ​​relative to the baseline.

[0684] Most participants entered the study with normal values ​​(i.e., within their respective reference ranges) for hematological, urinalytic, coagulation, blood chemistry, and urine chemistry parameters. No significant trend of change was observed between treatment groups. For some laboratory parameters, changes from normal values ​​at baseline to abnormal values ​​(Grade 1 [mild] or Grade 2 [moderate]) were observed during the study; however, these abnormal values ​​were considered clinically insignificant. Most changes were transient and resolved during the study period.

[0685] During the study, three subjects in the ALXN1210 SC group reported a transition to a grade 3 outlier. None of these transitions to grade 3 outliers were reported as adverse events (AEs).

[0686] First, one subject reported a decreased neutrophil count. This subject's neutrophil count at baseline was 3.77 × 10^9 / L. The neutrophil count assessed on day 43 was 0.95 × 10^9 / L (normal range: 2.0 to 7.5 × 10^9 / L). The neutrophil count returned to the normal range on day 57.

[0687] Two subjects reported elevated potassium levels (normal range: 3.5 to 5.1 mmol / L). In one subject with a baseline potassium level of 4.5 mmol / L, the potassium level assessed on day 150 was 6.1 mmol / L. Repeat potassium levels on the same day (out-of-plan visit) were within the normal range. In another subject with a baseline potassium level of 4.6 mmol / L, the potassium level assessed on day 90 was 6.4 mmol / L. This subject presented with abnormal potassium values ​​at screening (range 5.2 to 6.2 mmol / L during different screening visits) and at most study visits. The elevation in potassium levels was transient; values ​​recorded on days 150 and 200 were within the normal range.

[0688] There were no observable changes in vital sign measurements relative to baseline, and no clinically significant abnormalities were observed in the vital signs of the individual subjects during continuous observation.

[0689] Apart from those reported as adverse events (AEs), no physical examination results were clinically significant for any of the subjects. ECG or telemetry monitoring results showed no significant changes relative to baseline.

[0690] Variations in QT intervals were corrected for using Fridricia's formula (QTcF). In one subject in the placebo SC group, a mean QT interval >500 ms was observed at screening (510.0 ms) and on day 2 (508.7 ms), day 150 (516.6 ms), and day 200 (612.9 ms). At screening and on days 2, 150, and 200, the mean QTcF intervals for the same subject were 449.7 ms, 443.7 ms, 451.9 ms, and 501.3 ms, respectively. The increase in QT and QTcF intervals was considered clinically significant in this placebo-taking female subject. These changes were not reported as adverse events (AEs). No significant changes in mean QT and QTcF intervals relative to baseline were observed during the study period.

[0691] Infusion or injection site evaluations were performed within 15 minutes of the initial injection (SOI), and within ±15 minutes at 30 minutes, 2 hours, 4 hours, and 8 hours, as well as on day 2 (48 hours) and day 3 (72 hours, for a total of 6 evaluations). Indurations or reactions <1 cm were not considered adverse events (AEs) unless they persisted for more than 24 hours. In the ALIN1210 SC group, erythema was observed in 5 / 24 subjects 30 minutes after the initial injection (EOI), and in one subject, the smallest erythema (3 mm) was observed at the injection site in 2 / 4 of the subjects 2 hours after injection, with no erythema observed at the final time point. In the ALXN1210 SC group, the smallest induration or swelling (10 mm) was reported in 1 / 24 subjects 30 minutes after the EOI, with no induration or swelling observed at the final evaluation. However, these did not meet the protocol-defined criteria and were not considered AEs. Subjects rated pain at the infusion or injection site using the VAS (0 to 100 mm). For most infusions and injections, pain at the infusion site was rated as 0 mm at all assessments. Two subjects in the SC group reported transient pain of 3–5 mm on day 1, while three subjects in the IV group reported minimal pain (1–5 mm) after infusion.

[0692] 9. Safety conclusions

[0693] All subjects who received a single dose of the investigational drug (placebo SC, ALXN1210 SC, or ALXN1210 IV) were included in the safety group (N=42). Across the three treatment groups, 35 / 42 subjects (83.3%) experienced 75 treatment-associated adverse events (TEAEs). Only 3 / 75 TEAEs (4%) were considered ALXN1210-related, while 72 / 75 (96%) were considered unrelated to ALXN1210 treatment. All TEAEs were mild (Grade 1) and resolved during the study. Most TEAEs did not require any medication, and no non-pharmacological intervention was needed at any time. The most frequently reported TEAEs were nasopharyngitis (23 / 42 subjects, 54.8%) and headache (7 / 42 subjects, 16.7%).

[0694] No deaths or SAEs occurred during the study. Reported TEAEs did not lead to study drug discontinuation or subject withdrawal. Generally, no clinically significant changes were observed in laboratory parameters, vital signs, physical examinations, ECGs, or telemetry during the study or follow-up period. No clinical signs of hypersensitivity were observed during or after any single-dose SC injection or IV infusion. In subjects with positive ADA results, no clinical signs or symptoms associated with anaphylactic or hypersensitivity reactions were noted.

[0695] 10. Discussion and Overall Conclusion

[0696] The aim of this Phase 1 study was to evaluate the safety, tolerability, pharmacokinetics, PD, and immunogenicity of a single dose of ALXN1210 SC 400 mg compared to a single dose of ALXN1210 IV 400 mg or placebo SC in healthy subjects. A total of 42 subjects were randomized to receive the study drug: placebo SC (n=6); ALXN1210 SC (n=24); and ALXN1210 IV (n=12).

[0697] In healthy subjects, administration of ALXN1210 at a dose of 400 mg via the SC route was well tolerated. Based on AUC, the estimated GMR (SC / IV) of ALXN1210 SC was 60.4% (95% CI: 49.7, 73.3). The geometric mean t-values ​​after ALXN1210 SC and IV administrations were [not specified in the original text]. 1 / 2 The estimated values ​​were 31.3 days and 29.9 days, respectively. The severity and duration of PD response, assessed by free and total C5 serum concentrations and cRBC hemolysis, depended on the exposure.

[0698] In the ALXN1210 SC and ALXN1210 IV groups, 3 / 23 (13%) and 1 / 12 (8.3%) subjects, respectively, reported anti-antibody antibodies, with ADA positive titers ranging from <1.0 to 27. For SC and IV administration, the earliest post-dose responses were observed on day 29 and day 15, respectively. ADA cross-reacted with eculizumab in most ADA-positive samples following SC administration. In subjects showing positive ADA responses, no clinical signs or symptoms consistent with anaphylactic or hypersensitivity reactions (including anaphylaxis) were observed. Furthermore, no significant pharmacokinetic or PD effect on ALXN1210 could be identified.

[0699] No unexpected safety issues occurred in any treatment group during the study. No deaths or SAEs occurred during the study, and no subject experienced any TEAEs that led to discontinuation of the study drug or withdrawal from the study.

[0700] Sequence Summary

[0701]

[0702]

[0703]

[0704]

[0705] More specifically, this application provides the following:

[0706] 1. A stable aqueous solution comprising:

[0707] (a) An anti-C5 antibody at a concentration of about 100 mg / mL, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6;

[0708] (b) Approximately 50 mM phosphate buffer;

[0709] (c) Approximately 5% sucrose; and

[0710] (d) Approximately 25 mM arginine.

[0711] 2. A stable aqueous solution comprising:

[0712] (a) An anti-C5 antibody, wherein the anti-C5 antibody comprises a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 18, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6;

[0713] (b) Approximately 50 mM phosphate buffer;

[0714] (c) Approximately 5% sucrose;

[0715] (d) Approximately 0.05% polysorbate 80; and

[0716] (e) Approximately 25 mM arginine.

[0717] 3. The stable aqueous solution as described in item 1, further comprising a surfactant.

[0718] 4. A stable aqueous solution as described in item 3, wherein the surfactant is about 0.05% polysorbate 80.

[0719] 5. The stable aqueous solution as described in item 2, wherein the concentration of the anti-C5 antibody is [missing information].

[0720] Approximately 100 mg / mL.

[0721] 6. A stable aqueous solution as described in any of the preceding items, wherein the anti-C5 antibody further comprises a variant human Fc constant region that binds to the human neonatal Fc receptor (FcRn), wherein the variant human Fc CH3 constant region comprises Met-429-Leu and Asn-435-Ser substitutions at residues corresponding to methionine 428 and asparagine 434 of the natural human IgG Fc constant region, respectively, according to EU designations.

[0722] 7. A stable aqueous solution as described in any of the preceding claims, wherein the anti-C5 antibody comprises the heavy chain variable region depicted in SEQ ID NO: 12 and the light chain variable region depicted in SEQ ID NO: 8.

[0723] 8. A stable aqueous solution as described in any of the preceding items, wherein the anti-C5 antibody comprises the heavy chain constant region depicted in SEQ ID NO: 13.

[0724] 9. A stable aqueous solution as described in any of the preceding claims, wherein the anti-C5 antibody comprises a heavy chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 14 and a light chain polypeptide containing the amino acid sequence depicted in SEQ ID NO: 11.

[0725] 10. A stable aqueous solution as described in any of the preceding items, wherein the anti-C5 antibody is ALXN1210 (revozumab).

[0726] 11. A stable aqueous solution as described in any of the preceding items, wherein the pH of the solution is between 7.2 and 7.6.

[0727] 12. A stable aqueous solution as described in item 11, wherein the pH of the solution is 7.4.

[0728] 13. A stable aqueous solution as described in any of the preceding items, wherein the solution is sterile.

[0729] 14. A stable aqueous solution as described in any of the preceding items, wherein, as determined by SEC-HPLC, the anti-C5 antibody retains at least 97% monomeric during storage at 2°C to 8°C for at least six months.

[0730] 15. A stable aqueous solution as described in any of the preceding items, wherein, as determined by SEC-HPLC, the anti-C5 antibody retains at least 97% monomeric during storage at 2°C to 8°C for at least one year.

[0731] 16. A stable aqueous solution as described in any of the preceding items, wherein, as determined by SEC-HPLC, less than 3% of the anti-C5 antibody aggregates in the solution.

[0732] 17. A stable aqueous solution as described in any of the preceding items, wherein, as determined by SEC-HPLC, less than 2% of the anti-C5 antibody aggregates in the solution.

[0733] 18. A stable aqueous solution as described in any of the preceding items, wherein, as determined by SEC-HPLC, less than 1% of the anti-C5 antibody aggregates in the solution.

[0734] 19. A stable aqueous solution as described in any of the preceding items, wherein the anti-C5 antibody retains at least 90% of its C5 binding activity during storage at 2°C to 8°C for at least six months, compared to the anti-C5 antibody prior to storage.

[0735] 20. A stable aqueous solution as described in any of the preceding items, wherein the anti-C5 antibody retains at least 95% of its hemolytic activity during storage at 2°C to 8°C for at least six months, compared to the anti-C5 antibody prior to storage.

[0736] 21. A stable aqueous solution as described in any of the preceding items, wherein the solution is suitable for subcutaneous administration.

[0737] 22. A stable aqueous solution as described in any one of items 1-21, wherein the solution is suitable for intravenous administration.

[0738] 23. A method for producing a stable concentrated antibody solution, the solution comprising: an anti-C5 antibody at a concentration of 100 mg / mL, said anti-C5 antibody comprising a heavy chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 23, a heavy chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 19, a heavy chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 3, a light chain CDR1 containing the amino acid sequence depicted in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence depicted in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence depicted in SEQ ID NO: 6; 50 mM phosphate buffer; 5% sucrose; and 25 mM arginine, the method comprising:

[0739] i) Provide a first aqueous solution comprising the anti-C5 antibody, the first aqueous solution having a first formulation and containing no more than 10 mg / mL of the anti-C5 antibody;

[0740] ii) Percolate the first aqueous solution into a formulation containing 50 mM phosphate buffer, 5% sucrose, and 25 mM arginine at pH 7.4 to produce a second aqueous solution, wherein the second aqueous solution has a second formulation due to percolation; and

[0741] iii) Concentrate the second aqueous solution to produce a stable concentrated antibody solution containing 100 mg / mL of the anti-C5 antibody, 50 mM phosphate buffer, 5% sucrose and 25 mM arginine.

[0742] 24. A method of treating a human patient suffering from complement-related symptoms, comprising administering to the patient a stable aqueous solution as described in any one of items 1-21 in an amount effective in treating said complement-related symptoms.

[0743] 25. The method as described in item 23, wherein the complement-related symptoms are selected from 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, epidermolysis bullosa, recurrent miscarriage, traumatic brain injury, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / enterovascular disease, vasculitis, allergic purpuric nephritis, and generalized erythema. Lupus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Goyan's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki disease, venous air embolism, stent placement, coronary rotational atherectomy, restenosis after 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 I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpass syndrome, Diagos' disease, and catastrophic antiphospholipid syndrome.

[0744] 26. The method as described in item 24, wherein the complement-related symptom is atypical hemolytic uremic syndrome (aHUS).

[0745] 27. The method as described in item 24, wherein the complement-related symptom is paroxysmal nocturnal hemoglobinuria (PNH).

[0746] 28. The method of any one of items 24-26, wherein the stable aqueous solution is administered subcutaneously to the patient.

[0747] 29. The method of any one of items 24-26, wherein the stable aqueous solution is administered intravenously to the patient.

[0748] 30. A treatment kit comprising: (i) a stable aqueous solution according to any one of claims 1-21 and (ii) a tool for delivering said stable aqueous solution to a person.

[0749] 31. The treatment kit as described in item 28, wherein the tool is a syringe.

Claims

1. A stable aqueous solution comprising: (a) An anti-C5 antibody at a concentration of 70 mg / mL, wherein the anti-C5 antibody comprises: a heavy chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 14 and a light chain polypeptide comprising the amino acid sequence depicted in SEQ ID NO: 11; (b) 50 mM phosphate buffer; (c) 5% sucrose; and (d) 25 mM arginine; The pH of the solution is 7.2 to 7.

6.

2. The stable aqueous solution of claim 1, further comprising a surfactant.

3. The stable aqueous solution of claim 2, wherein the surfactant is 0.05% polysorbate 80.

4. The stable aqueous solution of claim 1, wherein the anti-C5 antibody is revozumab.

5. The stable aqueous solution of claim 1, wherein the pH of the solution is 7.

4.

6. A stable aqueous solution as claimed in any of the preceding claims, wherein: (a) The solution is sterile; (b) As determined by SEC-HPLC, the anti-C5 antibody retains at least 97% monomericity during storage at 2°C to 8°C for at least six months; (c) As determined by SEC-HPLC, less than 3% of the anti-C5 antibody aggregates in the solution; and / or (d) During storage at 2°C to 8°C for at least six months, the anti-C5 antibody: retains at least 90% of its C5 binding activity compared to the anti-C5 antibody prior to storage; and / or, retains at least 95% of its ability to inhibit hemolysis compared to the anti-C5 antibody prior to storage.

7. The stable aqueous solution of claim 6, wherein, as determined by SEC-HPLC, the anti-C5 antibody retains at least 97% monomeric during storage at 2°C to 8°C for at least one year.

8. The stable aqueous solution of claim 6, wherein, as determined by SEC-HPLC, less than 2% of the anti-C5 antibody aggregates in the solution.

9. The stable aqueous solution of claim 6, wherein, as determined by SEC-HPLC, less than 1% of the anti-C5 antibody aggregates in the solution.

10. A stable aqueous solution according to any one of claims 1 to 5, wherein the solution is suitable for subcutaneous administration.

11. The stable aqueous solution according to any one of claims 1 to 5, wherein the solution is in a cartridge, a pre-filled syringe, or a disposable pen.

12. A pharmaceutical composition for subcutaneous administration comprising a stable aqueous solution containing an anti-C5 antibody at a concentration of 70 mg / mL, wherein the anti-C5 antibody is revoizumab in a container at a concentration of 70 mg / mL and a pH of 7.4, and the stable aqueous solution further comprising 25 mM L-arginine, 0.05% polysorbate 80, 50 mM sodium phosphate, 5% sucrose, and water.

13. The pharmaceutical composition of claim 12, wherein the container is selected from vials, cartridges, pre-filled syringes, and disposable pens.

14. The pharmaceutical composition of claim 13, wherein the container is a cartridge.

15. Use of a stable aqueous solution of any one of claims 1 to 11 or a pharmaceutical composition of any one of claims 12 to 14 in the preparation of a medicament for treating complement-related symptoms in human patients, wherein the complement-related symptoms are atypical hemolytic uremic syndrome or paroxysmal nocturnal hemoglobinuria.

16. The use as described in claim 15, wherein the stable aqueous solution or the pharmaceutical composition is administered subcutaneously to the patient.

17. A therapeutic kit comprising: (i) a stable aqueous solution as described in any one of claims 1 to 10, or a pharmaceutical composition as described in any one of claims 12 to 14; (ii) A tool for delivering the stable aqueous solution or the pharmaceutical composition to a person.

18. The treatment kit of claim 17, wherein the tool is a delivery device.

19. The treatment kit of claim 18, wherein the delivery device is a disposable or reusable autoinjector, pen injector, patch injector, wearable injector, pre-filled injector, or removable injector infusion pump with a subcutaneous infusion kit.

20. The treatment kit of claim 19, wherein the delivery device is a removable syringe infusion pump with a subcutaneous infusion kit.

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