Pharmaceutical composition for treating or preventing transthyretin-mediated amyloidosis

Abstract

Transthyretin (TTR) is a soluble protein involved in thyroxine and retinol transport in the body. Under certain conditions, the TTR protein adopts a misfolded, misassembled, and / or aggregated TTR conformation, which can be toxic and lead to transthyretin-mediated amyloidosis (ATTR). Provided herein, inter alia, are compositions (e.g., pharmaceutical compositions) containing anti-TTR antibodies or antigenic fragments thereof, and related articles of manufacture. Further provided herein, inter alia, are methods for treating or preventing ATTR using the pharmaceutical compositions described herein.

Description

[Technical Field]

[0001] This application claims the benefit of priority to European Patent Application No. 22 207 645.7 and U.S. Provisional Patent Application No. 63 / 383,803, both filed November 15, 2022, and U.S. Provisional Patent Application No. 63 / 503,286, filed May 19, 2023.

[0002] The entire contents of the above-referenced application are incorporated herein by reference.

[0003] The present disclosure relates to pharmaceutical compositions for treating or preventing transthyretin-mediated amyloidosis (ATTR). [Background technology]

[0004] Transthyretin (TTR) is a soluble protein involved in thyroxine and retinol transport in the body. TTR is secreted into the blood by the liver and into the cerebrospinal fluid by the choroid plexus, and is also expressed in certain tissues such as pancreatic alpha cells and retinal epithelium.

[0005] Under certain conditions, which are poorly understood and may include acidic pH, oxidative stress, and local factors, the TTR protein can adopt misfolded, misassembled, and / or aggregated TTR conformations, which can become toxic and lead to transthyretin-mediated amyloidosis (ATTR).

[0006] There is a need for pharmaceutical formulations for preparing and administering antibodies (e.g., human anti-TTR antibodies) that target misfolded, misassembled, and / or aggregated TTR. Summary of the Invention

[0007] Provided herein, inter alia, are compositions (e.g., pharmaceutical compositions) and related articles of manufacture containing anti-transthyretin (TTR) antibodies or antigenic fragments thereof as drugs. Further provided herein, inter alia, are methods of treating or preventing transthyretin-mediated amyloidosis ATTR in a subject in need thereof using the pharmaceutical compositions described herein.

[0008] According to the present invention, a pharmaceutical formulation has been developed for a recombinant human monoclonal antibody specific for the disease-associated amyloidogenic form of TTR, characterized as ALXN2220, also known as NI006, at a concentration of about 25 mg / ml to about 150 mg / ml, typically about 50 mg / ml, essentially characterized by a pH of 5.3 to 6.3, preferably pH 5.8±0.1, typically containing a histidine buffer, sucrose, polysorbate, preferably polysorbate 80, and water for infusion / injection. The formulation of the present invention allows for long-term storage of the antibody in liquid form without the need for lyophilization, and its intravenous administration to subjects in need thereof. Indeed, the antibody formulation of the present invention has been successfully used in a first-in-human trial of NI006 in patients with amyloid transthyretin cardiomyopathy (ATTR-CM), where the formulation was administered at a dose of 0.3 to 60 mg per kilogram of body weight, without any drug-related or formulation-attributable adverse events. See ClinicalTrials.gov ID NCT04360434 and Garcia-Pavia et al., Phase 1 Trial of Antibody NI006 for Depletion of Cardiac Transthyretin Amyloid. N. Engl. J. Med. 389 (2023), 239-250, each of which is incorporated herein by reference.

[0009] International Application WO 2019 / 108689(A1) discloses the preparation of lyophilized formulations of two murine monoclonal anti-TTR antibodies, designated 9D5 and 14G8, and their humanized versions, with the most preferred formulation (Formulation F25) containing approximately 50 mg / ml of humanized antibody 14G8, 20 mM histidine, 240 mM sucrose, and 0.04% poloxamer 188 (PX188) at pH 6.0. See page 57, paragraph

[0269] of WO 2019 / 108689(A1). Notably, WO 2019 / 108689(A1) also describes the investigation of other buffers and excipients, including polysorbate 20 (PS20) and polysorbate (PS80), specifically excluding, for example, formulations containing sucrose and PS80 in combination. Furthermore, WO 2019 / 108689(A1) provides only two short-term experiments of the test formulation in the liquid state (1 week at 50°C and 2 weeks at 25°C), while the experiments of "long-term storage" at different temperatures (1 month and 3 months) are carried out with a freeze-dried formulation.

[0010] In contrast, the present invention generally relates to liquid formulations of anti-TTR antibodies with a long shelf life, which have the advantages of being ready to use without the need for reconstitution, reducing the risk of error since no reconstitution step is required, and more rapid administration, which is particularly beneficial in acute situations.

[0011] The solution to this goal, as characterized in the claims, is illustrated in the accompanying examples by preferred embodiments, in which the formulation consists essentially of the anti-TTR antibody ALXN2220 (also known as and referred to herein as NI006), a histidine buffer, and sucrose and polysorbate 80 (PS80) as excipients, having a pH of about 5.8 and a concentration of antibody of about 50 mg / ml or about 100 mg / ml.

[0012] In particular, a formulation of antibody at 50 mg / ml in 20 mM histidine, 6.5% or 8% (w / v) sucrose, and 0.03% (w / v) PS80 (pH 5.8) is identified in the Examples as a prime candidate for further development and clinical use, and therefore represents the most preferred embodiment of the invention, as well as equivalent formulations that maintain the physicochemical properties important to antibody stability and function as tested and validated in the accompanying Examples. In order to vary the concentrations of ingredients and / or alternative excipients in the preferred formulation, the following factors should be considered to ensure antibody stability and efficacy: Histidine: Acts as a buffer. Buffering capacity is related to histidine's ability to maintain pH near its pKa value. Because histidine's pKa is about 6.0, it is slightly less efficient at pH 5.8, but still functional as a buffer. Buffering capacity is also affected by the concentration of the buffer and the proximity of the pH to the pKa.

[0013] Sucrose: Stabilizes proteins during freeze / thaw cycles and increases osmolality. Sucrose contributes significantly to the osmolality of the solution, which is important for maintaining structural IgG antibodies.

[0014] High Concentration of Protein of Interest: Antibodies, like all proteins, have buffering capacity due to the presence of ionizable groups in their amino acid side chains, including the carboxyl groups of aspartic acid and glutamic acid, the amino groups of lysine and arginine, the imidazole groups of histidine, the hydroxyl groups of tyrosine, the thiol groups of cysteine, and the terminal amino and carboxyl groups.

[0015] pH 5.8: The pH is slightly lower than the pKa of histidine, which still provides a buffering effect. For example, if the original buffer system has a concentration of 20 mM histidine and a pH of 5.8, the ratio of conjugate base to acid can be calculated using the Henderson-Hasselbalch equation. To maintain the pH, the concentrations of both the protonated (HA) and deprotonated (A-) forms can be doubled to 40 mM. Since changing the total concentration of the buffer affects the ionic strength and osmolality of the solution, which can affect the stability and solubility of proteins, the concentration and / or nature of the osmolality agent, in this case sucrose, can be adjusted, and the corresponding formulations can be tested according to the Examples.

[0016] The parent antibody of Ni006 was first described in WO 2015 / 092077(A1) (designated antibody NI-301.37F1) and Michalon et al., Nat. Commun. 12 (2021), 3142 (designated antibody NI301A). As disclosed in WO 2015 / 092077(A1), NI006 (NI-301.37F1) is characterized by its binding, inter alia, to aggregated human wild-type transthyretin (wtATTR), as shown in Figures 2-4 and 7, described in Examples 3-6, and further described in the last paragraph of page 46. Furthermore, WO 2015 / 092077(A1) discloses that NI006 (NI-301.37F1) does not bind to human native transthyretin (TTR) monomers or dimers, as shown in Example 5 and Figure 4. This binding profile is advantageous because it selectively binds to aggregated wtTTR and therefore clearly allows for the treatment of not only hereditary transthyretin amyloidosis (hATTR) with polyneuropathy caused by mutations in the gene encoding TTR (formerly known as familial amyloid polyneuropathy, or FAP), but also wild-type transthyretin amyloidosis (wtATTR), also known as senile systemic amyloidosis (SSA). Furthermore, the antibody does not risk interfering with the assembly of native monomers into the physiological tetramer. Thus, in a preferred embodiment of the pharmaceutical antibody formulation of the invention, the antibody is ALXN2220 / NI006 or an equivalent antibody that has substantially the TTR binding profile of NI006 and is preferably of human origin. For example, WO 2015 / 092077(A1) discloses two further human antibodies that exhibit the mentioned binding profile, namely, antibodies NI-301.59F1 and NI-301.35G11, as well as two human antibodies NI-301.28B3 and NI301.12D3, which have substantially the same epitope as NI006 (NI-301.37F7).

[0017] While human-derived antibodies are particularly preferred, for example because they are inherently less susceptible to anti-drug antibody (ADA) responses, humanized antibodies and human-sequence monoclonal antibodies derived from animals such as mice are included in the formulations of the present invention. In addition, as previously noted, the formulations have been proven safe and effective. See ClinicalTrials.gov ID NCT04360434 and Garcia-Pavia et al. (2023) (supra).

[0018] As illustrated in the accompanying examples, several stability studies performed with the formulations and pharmaceutical compositions of the invention demonstrate that the antibodies remain stable under a variety of conditions, e.g., at 40±2°C and 75±5% relative humidity (RH) for at least 1 month (stress stability study); at 25±2°C / 60±5% RH for at least 6 months (accelerated stability study); and at 5±3°C for at least 12-18 months (long-term stability study).

[0019] Accordingly, the present invention relates to a pharmaceutical composition (also referred to herein as a pharmaceutical formulation) comprising a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof capable of binding to mutated, misfolded, misassembled, and / or aggregated TTR species, particularly aggregated human TTR species, and / or fragments thereof, and which does not substantially recognize physiological TTR species, wherein the pharmaceutical composition comprises one or more of sucrose, polysorbate, preferably polysorbate 80, and a polar excipient (e.g., L-histidine and / or histidine, such as L-histidine monohydrochloride, or a pharmaceutically acceptable salt thereof), and wherein the antibody remains stable at 40±2°C and 75±5% RH for at least 1 week, preferably at least up to 1 month; at 25±2°C / 60±5% RH for at least 1 month, preferably at least 6 months; and / or at 5±3°C for at least 1 month, preferably at least 12 months, more preferably at least 18 months. In a preferred embodiment, the pharmaceutical composition is an aqueous composition, also referred to as a liquid formulation, and therefore also comprises water for injection.

[0020] The polar excipient included in the pharmaceutical composition of the present invention is also referred to as a buffer system, preferably a histidine buffer (eg, composed of L-histidine and L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof).

[0021] In a preferred embodiment, the polar excipient (e.g., L-histidine and / or histidine such as L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof) is present in the pharmaceutical composition of the present invention at a concentration of about 1 mM to about 100 mM (e.g., about 1 mM to about 50 mM, e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM, e.g., about 10 mM to about 30 mM, e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM). , about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, e.g., about 50 mM to about 100 mM, e.g., about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM), and most preferably, the polar excipient comprises about 20 mM histidine (e.g., provided by L-histidine and / or L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof, preferably 1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride).

[0022] Additionally, or alternatively, the pharmaceutical compositions of the present invention contain about 6% to about 9% (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9%) sucrose by weight per unit volume (w / v).

[0023] Preferably, the pharmaceutical composition of the present invention contains about 6% to about 7% (w / v) sucrose (e.g., about 6%, about 6.1%, about 6.2%, about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%, about 6.9%, or about 7%), and more preferably, the pharmaceutical composition of the present invention contains about 6.5% (w / v) sucrose.

[0024] Alternatively, the pharmaceutical compositions of the present invention contain about 7.5% to about 8.5% (e.g., about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8%, about 8.1%, about 8.2%, about 8.3%, about 8.4%, or about 8.5%) (w / v) sucrose, and most preferably, the pharmaceutical compositions of the present invention contain about 8% (w / v) sucrose.

[0025] Furthermore, in any of the aforementioned embodiments, the pharmaceutical composition of the present invention comprises about 0.001% to about 0.1% (w / v) (e.g., about 0.001% to about 0.01%, e.g., about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, or 0.01%, e.g., about 0.01% to about 0.1%, e.g., about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%) of a polysorbate, preferably polysorbate 80.

[0026] More preferably, the pharmaceutical composition of the present invention contains about 0.01% to about 0.05% (w / v) (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, or about 0.05%) of polysorbate 80, and most preferably, the pharmaceutical composition of the present invention contains about 0.03% (w / v) of polysorbate 80.

[0027] In a preferred embodiment, the pharmaceutical composition of the present invention has a pH of about 5.3 to about 6.3 (e.g., about 5.3 to about 5.8, e.g., about 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8, e.g., about 5.5 to about 6.0, e.g., about 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0, e.g., about 5.8 to about 6.3, e.g., about 5.8, 5.9, 6.0, 6.1, 6.2, or 6.3).

[0028] Most preferably, the pharmaceutical compositions of the present invention have a pH of about 5.8.

[0029] In principle, the antibody contained in the pharmaceutical composition can be any anti-TTR antibody that recognizes the amyloidogenic form of TTR, i.e., aggregated TTR species, preferably human aggregated TTR, but does not bind to physiological TTR species. In a particularly preferred embodiment, the anti-TTR antibody used in the pharmaceutical composition of the present invention is NI006 / ALXN2220 or an equivalent antibody derived from the human antibody NI-301.37F1 characterized in WO 2015 / 092077(A1) and Michalon et al., Nat Commun. 12 (2021), 3142. See also above.

[0030] Therefore, in a preferred embodiment, the anti-TTR antibody or antigen-binding fragment thereof contained in the pharmaceutical composition of the present invention comprises a heavy chain variable region (VH) having three complementarity-determining regions (CDRs) set forth in SEQ ID NOS: 1 to 3 and a light chain variable region (VL) having three CDRs set forth in SEQ ID NOS: 4 to 6.

[0031] In some embodiments, the VH region comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to SEQ ID NO:7, and the VL region comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to SEQ ID NO:8.

[0032] In some embodiments, the VH region comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to SEQ ID NO: 11, and the VL region comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity) to SEQ ID NO: 12.

[0033] Preferably, the VH region comprises the amino acid sequence of SEQ ID NO: 7 and the VL region comprises the amino acid sequence of SEQ ID NO: 8, or the VH region comprises the amino acid sequence of SEQ ID NO: 11 and the VL region comprises the amino acid sequence of SEQ ID NO: 12.

[0034] In some embodiments, the VH region of the anti-TTR antibody or antigen-binding fragment thereof comprises one or more amino acid sequences having about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3. The VL region of the anti-TTR antibody or antigen-binding fragment thereof comprises one or more CDR sequences comprising an amino acid sequence having about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, and / or SEQ ID NO:6. In some embodiments, the VH region of the anti-TTR antibody or antigen-binding fragment thereof comprises one or more CDR sequences having an amino acid sequence containing one, two, or three mismatches to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3, and the VL region of the anti-TTR antibody or antigen-binding fragment thereof comprises one or more CDR sequences having an amino acid sequence containing one, two, or three mismatches to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:5, and / or SEQ ID NO:6.

[0035] To avoid the generation of "anti-drug antibodies" (ADAs) by subjects administered the antibodies or antigen-binding fragments thereof described herein, the antibodies are preferably human or humanized, typically human IgG, most preferably human IgG1. In a preferred embodiment, the antibody is of the human IgG1m3 allotype. Accordingly, the anti-TTR antibody used in the pharmaceutical compositions of the present invention is preferably NI006 / ALXN2220, or an equivalent antibody derived from the human antibody NI-301.37F1 characterized in WO 2015 / 092077(A1) and Michalon et al., Nat Commun. 12 (2021), 3142. Antibody NI006 / ALXN2220 is a fully human IgG1m3 allotype antibody and therefore comprises the human constant heavy chain (HC) amino acid sequence present in SEQ ID NO: 9 and the corresponding human constant light chain (LC), exemplified here by a kappa light chain in SEQ ID NO: 10. As explained further below, IgG antibodies are organized as a tetramer consisting of a HC and two light LC chains linked by disulfide bridges.

[0036] The antibody NI006 / ALXN2220 can be produced in Chinese hamster ovary (CHO) cells. CHO cells are the most widely used mammalian cells for the production of recombinant monoclonal antibodies due to their ability to perform post-translational modifications (PTMs) on antibody molecules (which typically also occur in humans). Through genetic engineering by mutagenesis, different CHO daughter cells with improved qualities have been established. Among these variants are CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Thus, in one embodiment, the antibody used in the pharmaceutical formulation of the present invention is produced in CHO cells, preferably CHO-K1 cells, and purified from the cell culture medium for further use.

[0037] The major PTMs identified in antibody NI006 / ALXN2220 are modification of the N-terminal glutamine to pyroglutamic acid in the HC, loss of the C-terminal lysine, and N-glycosylation, as shown in Example 3. In this context, an N-glycosylation site was identified at position Asn300 (HC N300, SEQ ID NO: 9).

[0038] Thus, in one embodiment, the heavy chain of the anti-TTR antibody present in the pharmaceutical composition of the present invention is missing a C-terminal lysine, i.e., the antibody has undergone C-terminal lysine clipping. In particular, the C-terminal lysine shown in SEQ ID NO: 9 is clipped from the heavy chain of the antibody, preferably from each heavy chain of the antibody. The sequence described above, i.e., the sequence of the heavy chain with the C-terminal lysine clipped, is set forth in SEQ ID NO: 13. Alternatively, the N-terminal glutamine is modified as pyroglutamic acid, i.e., the heavy chain of the antibody shown in SEQ ID NO: 9 has undergone N-terminal glutaminyl cyclization. The sequence described above, i.e., the sequence of the heavy chain containing cyclic pyroglutamic acid but without N-terminal glutamine, is set forth in SEQ ID NO: 14.

[0039] Alternatively, the heavy chain of the anti-TTR antibody present in the pharmaceutical composition of the present invention lacks the C-terminal lysine and the N-terminal glutamine is modified to pyroglutamic acid. The sequence described above, i.e., the sequence of the heavy chain from which the C-terminal lysine has been truncated, contains cyclic pyroglutamic acid, and does not contain the N-terminal glutamine, is set forth in SEQ ID NO: 15.

[0040] Additionally or alternatively, the antibody is glycosylated, particularly N-glycosylated, more particularly the heavy chain of the antibody is glycosylated, even more particularly glycosylated at N300 of the heavy chain.

[0041] In a preferred embodiment, the anti-TTR antibody present in the pharmaceutical composition of the present invention lacks a C-terminal lysine, has an N-terminal glutamine modified as pyroglutamic acid, and contains at least one N-glycosylation site. Thus, in a preferred embodiment, the pharmaceutical formulation of the present invention comprises an anti-TTR antibody as defined hereinabove, wherein the antibody is composed of two heavy chains having SEQ ID NO:9 and two light chains having SEQ ID NO:10, wherein the N-terminal glutamine in the heavy chain is modified as pyroglutamic acid, the C-terminal lysine is missing, and the heavy chain is N-glycosylated. In other words, in a preferred embodiment, the pharmaceutical formulation of the present invention comprises an anti-TTR antibody as defined hereinabove, wherein the antibody is composed of two heavy chains having SEQ ID NO:15 and two light chains having SEQ ID NO:10, wherein the heavy chain is N-glycosylated.

[0042] As shown in Example 3, about 99% to 100% of the antibodies present in a sample of a typical antibody formulation have an N-terminal pyroglutamic acid in their heavy chains, and about 96% of the antibodies have a loss of a C-terminal lysine. Thus, in one embodiment, about 99% of the antibodies in a formulation of the invention have heavy chains in which an N-terminal pyroglutamic acid has been modified from an N-terminal glutamine, and / or about 96% of the antibodies have a loss of a C-terminal lysine.

[0043] In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof is present in the pharmaceutical composition of the invention at a concentration of about 1 mg / mL to about 500 mg / mL (e.g., about 1 mg / mL to about 100 mg / mL, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, or about 100 mg / mL, e.g., about 100 mg / mL to about 200 mg / mL, e.g., about 100 mg / mL, about 110 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL, or about 200 mg / mL, e.g., about 200 mg / mL to about 30 The compound is present at a concentration of about 0 mg / mL, for example, about 200 mg / mL, about 210 mg / mL, about 225 mg / mL, about 250 mg / mL, about 275 mg / mL, or about 300 mg / mL, for example, about 300 mg / mL to about 400 mg / mL, for example, about 300 mg / mL, about 310 mg / mL, about 325 mg / mL, about 350 mg / mL, about 375 mg / mL, or about 400 mg / mL, for example, about 400 mg / mL to about 500 mg / mL, for example, about 400 mg / mL, about 410 mg / mL, about 425 mg / mL, about 450 mg / mL, about 475 mg / mL, or about 500 mg / mL).

[0044] In a preferred embodiment, the anti-TTR antibody or antigen-binding fragment thereof is present in the pharmaceutical composition of the present invention at a concentration of about 1 mg / mL to about 150 mg / mL (e.g., about 1 mg / mL to about 20 mg / mL, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, or about 20 mg / mL, e.g., about 20 mg / mL to about 40 mg / mL, e.g., about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, or about 40 mg / mL, about 40 mg / mL to about 60 mg / mL, e.g., about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 5 2 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, or about 60 mg / mL, for example, about 60 mg / mL to about 80 mg / mL, for example, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, or about 80 mg / mL, for example, about 80 mg / mL to about 1 The compound is present at a concentration of about 100 mg / mL, e.g., about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, about 105 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 125 mg / mL, about 130 mg / mL, about 135 mg / mL, about 140 mg / mL, about 145 mg / mL, or about 150 mg / mL. In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof is present in the pharmaceutical composition at a concentration of about 1 mg / mL to about 150 mg / mL and in a volume of about 0.1 mL to about 100 mL (e.g., the anti-TTR antibody or antigen-binding fragment thereof is present in the pharmaceutical composition at a concentration of about 50 mg / mL, about 100 mg / mL, about 125 mg / mL, or about 150 mg / mL and in a volume of about 2 mL or 20 mL).

[0045] Even more preferably, the anti-TTR antibody or antigen-binding fragment thereof is present in the pharmaceutical composition at a concentration of about 50 mg / mL, about 100 mg / mL, about 125 mg / mL, or about 150 mg / mL, most preferably about 50 mg / mL.

[0046] As described above, the anti-TTR antibody or antigen-binding fragment thereof included in the pharmaceutical composition of the present invention is preferably a human IgG1 antibody. For example, the human IgG1 antibody has a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 or 11 and a light chain variable region having the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof does not induce an anti-drug antibody (ADA) response.

[0047] In some embodiments, the pharmaceutical composition of the present invention is administered in a volume of about 0.1 mL to about 100 mL (e.g., about 0.1 mL to about 20 mL, e.g., about 1 mL, about 2 mL, about 2.25 mL, about 3 mL, about 5 mL, about 7 mL, about 10 mL, about 12 mL, about 15 mL, about 17 mL, or about 20 mL, e.g., about 20 mL to about 40 mL, e.g., about 20 mL, about 22.5 mL, about 25 mL, about 30 mL, about 35 mL, or about 40 mL, e.g., about 40 mL to about 60 mL, e.g., about 40 mL, about 45 mL, about 50 mL, about 55 mL, or about 60 mL, e.g., about 60 mL to about 80 mL, e.g., about 60 mL, about 65 mL, about 70 mL, about 75 mL, or about 80 mL, e.g., about 80 mL to about 100 mL, e.g., about 80 mL, about 85 mL, about 90 mL, about 95 mL, or about 100 mL).

[0048] Preferably, the pharmaceutical composition of the present invention is administered in a volume of about 1 mL to about 10 mL (e.g., about 1 mL to about 2 mL, e.g., about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9 mL, or about 2 mL, e.g., about 1.5 mL to about 2.5 mL, e.g., about 1.5 mL, about 1.6 mL, about 1.7 mL, about 1.8 mL, about 1.9 mL, about 2 mL, about 2.1 mL, about 2.2 mL, about 2.3 mL, about 2.4 mL, or about 2. 5 mL, for example, about 2 to about 2.25 mL, for example, about 2.1 mL, about 2.15 mL, about 2.2 mL, or about 2.25 mL, for example, about 2 mL to about 5 mL, for example, about 2 mL, about 2.5 mL, about 3 mL, about 3.5 mL, about 4 mL, about 4.5 mL, or about 5 mL, for example, about 5 mL to about 10 mL, for example, about 5 mL, about 5.5 mL, about 6 mL, about 6.5 mL, about 7 mL, about 7.5 mL, about 8 mL, about 8.5 mL, about 9 mL, about 9.5 mL, or about 10 mL).

[0049] More preferably, the pharmaceutical composition of the present invention is present in a volume of about 2 mL to about 20 mL (e.g., about 2 mL, about 2.25 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, or about 20 mL).

[0050] Alternatively, the pharmaceutical composition of the present invention is present in a volume of about 20 mL to about 25 mL (e.g., about 20 mL, about 20.5 mL, about 22 mL, about 22.5 mL, about 23 mL, about 23.5 mL, about 24 mL, about 24.5 mL, or about 25 mL).

[0051] Most preferably, the pharmaceutical composition of the present invention is present in a volume of about 2 mL or about 20 mL.

[0052] Additionally, the antibody formulation's compatibility with clinically-used materials was evaluated. Specifically, compatibility with polyvinyl chloride IV bags, IV lines, and filters, as well as compatibility with polyvinyl chloride syringes and corresponding non-polyvinyl chloride materials, when glucose or saline was used as a diluent, was evaluated. Three concentrations, 1 mg / mL, 20 mg / mL, and 50 mg / mL, were evaluated, and the data provided in Tables 52 and 53 in Example 2 indicate that the formulation was generally compatible with the clinically-used materials evaluated. Notably, visible particles were observed in the saline group, indicating that NI006 / ALXN2220 was more unstable in saline. The data also show that no substantial changes were observed in appearance, protein concentration, subvisible particles, SEC-HPLC, or ELISA binding assays when diluted in glucose solution. Therefore, NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL was stable for 24 hours at 2–8°C, followed by 6 hours at 25°C (a total of 30 hours), demonstrating compatibility with the clinically-used materials evaluated.

[0053] The most common route for therapeutic administration of monoclonal antibodies is intravenous (IV) infusion, which is preferred because it allows for direct delivery of the antibody into the bloodstream, ensuring immediate distribution throughout the body and allowing for precise dosing.

[0054] Intravenous administration is particularly important for monoclonal antibodies due to their large molecular size, which generally prevents them from being effectively absorbed through the intestine or skin. This means that oral or transdermal delivery methods are not suitable for these types of drugs. Furthermore, IV administration avoids first-pass metabolism in the liver, which can significantly alter the efficacy and safety profile of the drug.

[0055] Therefore, the formulations of the present invention have been developed specifically for IV administration. Thus, in a preferred embodiment, the formulations of the present invention are suitable for intravenous administration.

[0056] In some embodiments, the pharmaceutical composition has not been reconstituted from a lyophilized anti-TTR antibody or antigen-binding fragment thereof and / or has not been further lyophilized.

[0057] In the formulations of the present invention, sucrose is used as an antibody tonicity adjuster and additional stabilizer, so there is no need for NaCl, especially when the primary purpose of NaCl is to stabilize the protein, which sucrose can achieve without increasing the ionic strength of the solution. Thus, it is preferred that the pharmaceutical compositions of the present invention are essentially free of sodium chloride.

[0058] Additionally, or alternatively, the pharmaceutical compositions of the present invention are essentially free of (or, for example, completely devoid of) poloxamer.

[0059] In some embodiments, the pharmaceutical compositions of the invention are characterized by one, two, three, or all four stability criteria a)-(d) in any combination: (a) a main peak decrease of less than 1% by weight of the antibody under heat stress conditions at about 40° C. for 4 weeks and / or at about 25° C. for 12 weeks, as measured by size exclusion chromatography (SEC)-HPLC analysis; (b) the pharmaceutical composition exhibits an acidic species content of the anti-TTR antibody of less than 42.5% under heat stress conditions at about 40° C. for 2 weeks, as measured by capillary isoelectric focusing (cIEF). (c) the pharmaceutical composition does not exhibit a substantial change in the acidic species content of the anti-TTR antibody after three cycles of freeze-thawing (from about -70°C to room temperature) as measured by capillary isoelectric focusing (cIEF); and / or (d) the anti-TTR antibody retains at least 80% of its binding ability to TTR protein after 4 weeks of storage at about 40°C and / or at least 70% of its binding ability to TTR protein after 12 weeks of storage at about 25°C as measured, for example, by ELISA and compared to a control (e.g., no long-term storage).

[0060] In some embodiments, the pharmaceutical composition exhibits a main peak ≧50.0%, an acidic peak ≦40.0%, and a basic peak ≦15.0%, e.g., as measured by cIEF; a main peak (monomer) ≧95.0% and high molecular weight species (HMWS) ≦5.0%, e.g., as measured by size exclusion chromatography (SEC)-HPLC analysis; a pH of 5.8±0.5; an osmolality of ≧240 mOsm / Kg; and an antibody concentration of 50±5.0 mg / mL.

[0061] Additionally, the pharmaceutical composition of the present invention may be characterized by one, two, three, or all four stability criteria (i)-(iv) in any combination: (i) the main peak decline (representing monomer content) during long-term storage at about 40° C. for 1 month, or at about 25° C. for 6 months, or at about 5° C. for 18 months, as measured by SEC-HPLC, is less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2%; (ii) the pharmaceutical composition declines during long-term storage at about 40° C. for 2 weeks, or at about 25° C. for 3 months, as measured by cIEF. (iii) the pharmaceutical composition exhibits an anti-TTR antibody acidic species content of less than about 40% under long-term storage conditions at about 5°C for 12 or 18 months, as measured by cIEF; and / or (iv) the anti-TTR antibody retains at least 80%, preferably at least 90%, of its binding ability to TTR protein, as measured by ELISA, after 6 months of storage at about 25°C or after 12 or 18 months of storage at about 5°C, compared to a control (e.g., without long-term storage). Thus, the pharmaceutical composition can be referred to as a stable formulation. As described in Example 2, the shelf life of pharmaceuticals is currently set at 24 months at 5±3°C protected from light. Thus, in one embodiment, the pharmaceutical composition, i.e., the formulation, has a shelf life of 24 months at 2-8°C when protected from light.

[0062] Additionally or alternatively, the pharmaceutical composition of the present invention has an osmolality of 240 mOsm / Kg or greater and comprises sucrose, and optionally a surfactant.

[0063] In some embodiments, the pharmaceutical compositions of the present invention are sterile, particularly for pharmaceutical use.

[0064] In some embodiments, the pharmaceutical compositions of the present invention are stable upon freezing and thawing.

[0065] In some embodiments, the pharmaceutical compositions of the invention are present in a vial, for example, a 2 mL or 20 mL Type I clear glass vial.

[0066] In a preferred embodiment, the vial contains an overfill of about 12.5% ​​volume, or a total volume of about 2.25 mL or 22.5 mL of an anti-TTR antibody or antigen-binding fragment thereof.

[0067] In a preferred embodiment, the pharmaceutical composition of the present invention is a stable formulation and comprises an anti-TTR antibody as defined hereinabove, comprising an immunoglobulin heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 9, 13, 14, or 15, preferably SEQ ID NO: 9 or 15, and most preferably SEQ ID NO: 15 (i.e., SEQ ID NO: 9 having the PTM set forth above), at a concentration of about 25 mg / ml to about 150 mg / ml, and an immunoglobulin light chain (LC) comprising the amino acid sequence of SEQ ID NO: 10; histidine at a concentration of about 20 mM; sucrose at a concentration of about 50 mg / ml to about 80 mg / ml; polysorbate 80 (PS) at a concentration of about 0.01% (w / v) to about 0.1% (w / v); and water for injection, wherein the pharmaceutical composition has a pH of about 5.3 to about 6.3.

[0068] In an overall preferred embodiment, the pharmaceutical composition of the invention comprises 8% (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine (e.g., L-histidine), pH 5.8, and a volume of about or exactly 2.0 mL, wherein the anti-TTR antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region having the amino acid sequence of SEQ ID NO:7 or 11, preferably SEQ ID NO:11, and a light chain variable (VL) region having the amino acid sequence of SEQ ID NO:8; preferably, the antibody is a fully human IgG1m3 allotype antibody and therefore comprises a human heavy chain (HC) amino acid sequence present in SEQ ID NO:9 and a human light chain (LC), here a kappa light chain exemplified by SEQ ID NO:10, preferably together with the PTMs described above, i.e., preferably the HC amino acid sequence present in SEQ ID NO:15. Preferably, the pharmaceutical composition comprises the antibody at a concentration of 50 mg / mL or 100 mg / mL, most preferably 50 mg / mL.

[0069] Alternatively, the pharmaceutical composition comprises 6.5% (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine (e.g., L-histidine), pH 5.8, and a volume of about or exactly 2.0 mL, wherein the anti-TTR antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:7 or 11, preferably SEQ ID NO:11, and a VL region having the amino acid sequence of SEQ ID NO:8; preferably, the antibody is a fully human IgG1m3 allotype antibody and therefore comprises a human heavy chain (HC) amino acid sequence present in SEQ ID NO:9 and a human light chain (LC), here a kappa light chain exemplified by SEQ ID NO:10, preferably together with the PTMs described above, i.e., preferably the HC amino acid sequence present in SEQ ID NO:15. Preferably, the pharmaceutical composition comprises the antibody at a concentration of 50 mg / mL or 100 mg / mL, most preferably 50 mg / mL.

[0070] The present invention further relates to a kit comprising the pharmaceutical composition of the present invention and instructions for its use.

[0071] In some embodiments, the kit is for use in treating or preventing transthyretin-mediated amyloidosis ATTR in a human subject.

[0072] The present invention further relates to a method of treating ATTR in a human subject, comprising administering to the human subject a pharmaceutical composition of the present invention.

[0073] The present invention further relates to a method of treating ATTR amyloidosis with cardiomyopathy (ATTR-CM) or sporadic wild-type ATTR-CM (e.g., a wild-type ATTR gene encoding the TTR protein that forms deposits in the heart) in a human subject, comprising administering to the human subject a pharmaceutical composition of the present invention.

[0074] The present invention further relates to a pharmaceutical composition comprising a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL to about 150 mg / mL (e.g., 50 mg / mL, about 60 mg / mL, 70 mg / mL, about 80 mg / mL, 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL), a histidine (e.g., L-histidine) buffer at a pH of about 5.8, 6.5% or 8.0% w / v sucrose, and 0.03% w / v polysorbate 80. The anti-TTR antibody or antigen-binding fragment thereof can bind to mutated, misfolded, misassembled, and / or aggregated TTR species and / or fragments thereof, and does not substantially recognize physiological TTR species.

[0075] The present invention further provides a medicament comprising the pharmaceutical composition of the present invention, e.g., the medicament is for use in a method of treating ATTR in a subject in need thereof, e.g., for use in treating a subject with ATTR amyloidosis with cardiomyopathy (ATTR-CM), such as sporadic WT-ATTR-CM.

[0076] The present invention further relates to a pharmaceutical container, preferably a sterile container, comprising a pharmaceutical composition of the invention or a medicament of the invention, for example, the container is a disposable glass vial containing about 100 mg of antibody at a concentration of about 50±5 mg / mL to about 150 mg / mL (e.g., about 50 mg / mL, about 60 mg / mL, 70 mg / mL, about 80 mg / mL, 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL, e.g., about 100 mg / mL), and optionally the vial comprises an overfill of approximately 10% or 12.5% ​​volume.

[0077] In a preferred embodiment, the pharmaceutical composition of the present invention is ready to use, with or without dilution, for administration to a subject in need thereof, such as by intravenous infusion. Preferably, the pharmaceutical composition is a sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles, for intravenous use by infusion after dilution.

[0078] Preferably, the pharmaceutical composition is diluted in a solution containing glucose or a polymer thereof (e.g., dextran) before injection. Thus, preferably, glucose or a polymer thereof (e.g., dextran) is used as a diluent, and the pharmaceutical composition is added to the diluent before injection. In some embodiments, the concentration of glucose or a polymer thereof is 5% (w / v). In some embodiments, the antibody in the pharmaceutical composition is not diluted to less than 1 mg / mL.

[0079] In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof has a concentration of about 1 mg / mL to about 50 mg / mL (e.g., about 1 mg / mL to about 42 mg / mL, about 1 mg / mL to about 30 mg / mL, about 1 mg / mL to about 20 mg / mL, or about 1 mg / mL to about 10 mg / mL, e.g., about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, or about 21 mg / mL).

[0049] The compound is administered to a subject in an aqueous formulation at a dilution of about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, or about 50 mg / mL.

[0080] The present invention further relates to a method for treating ATTR (e.g., ATTR-CM or WT-ATTR-CM) in a human subject by administering to the subject a pharmaceutical composition of the present invention. In one embodiment, the method comprises administering to the subject a pharmaceutical composition at a dose providing about 1 to 60 mg / kg (e.g., about 10 to 60 mg / kg, about 30 to 60 mg / kg, about 30 mg / kg, or about 60 mg / kg) of human anti-TTR antibody, or about 3000 to 7500 mg (e.g., about 4000 to 7000 mg, about 3000 to 6000 mg, or about 4000 to 5000 mg, e.g., about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, about 6000 mg, about 6500 mg, about 7000 mg, or about 7500 mg) of human anti-TTR antibody. In one embodiment, about 3000 mg of a human anti-TTR antibody or antigen-binding fragment thereof is administered to a human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or antigen-binding fragment thereof is administered to a human subject.

[0081] The present invention further relates to methods of treating ATTR (e.g., ATTR-CM or WT-ATTR-CM) in a human subject by administering to the subject a pharmaceutical composition of the present invention. In some embodiments, the method comprises administering to the subject a pharmaceutical composition in a dose providing about 30-60 mg / kg (e.g., about 1-60 mg / kg (e.g., about 10-60 mg / kg, about 30-60 mg / kg, about 30 mg / kg, or about 60 mg / kg)) of human anti-TTR antibody, or about 3000-7500 mg (e.g., about 4000-7000 mg, about 3000-6000 mg, or about 4000-5000 mg, e.g., about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, about 6000 mg, about 6500 mg, about 7000 mg, or about 7500 mg) of human anti-TTR antibody. In some embodiments, about 3000 mg of a human anti-TTR antibody or antigen-binding fragment thereof is administered to the human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or antigen-binding fragment thereof is administered to a human subject.

[0082] The present invention further relates to pharmaceutical compositions of the present invention for treating ATTR (e.g., ATTR-CM or WT-ATTR-CM) in a subject in need thereof. In certain embodiments, the pharmaceutical compositions are for administration at a dose providing about 1 to 60 mg / kg (e.g., about 10 to 60 mg / kg, about 30 to 60 mg / kg, about 30 mg / kg, or about 60 mg / kg) of human anti-TTR antibody, or about 3000 to 7500 mg (e.g., about 4000 to 7000 mg, about 3000 to 6000 mg, or about 4000 to 5000 mg, e.g., about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, about 6000 mg, about 6500 mg, about 7000 mg, or about 7500 mg) of human anti-TTR antibody. In one embodiment, about 3000 mg of a human anti-TTR antibody or antigen-binding fragment thereof is used to treat a human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or antigen-binding fragment thereof is used to treat a human subject.

[0083] The present invention further relates to pharmaceutical compositions of the present invention for treating ATTR (e.g., ATTR-CM or WT-ATTR-CM) in a subject in need of such treatment. In certain embodiments, the pharmaceutical compositions are for administration at a dose providing about 1 to 60 mg / kg (e.g., about 10 to 60 mg / kg, about 30 to 60 mg / kg, about 30 mg / kg, or about 60 mg / kg) of human anti-TTR antibody, or about 3000 to 7500 mg (e.g., about 4000 to 7000 mg, about 3000 to 6000 mg, or about 4000 to 5000 mg, e.g., about 3000 mg, about 3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, about 6000 mg, about 6500 mg, about 7000 mg, or about 7500 mg) of human anti-TTR antibody. In one embodiment, about 3000 mg of a human anti-TTR antibody or antigen-binding fragment thereof is used to treat a human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or antigen-binding fragment thereof is used to treat a human subject.

[0084] The present invention further relates to the use of a pharmaceutical composition in the manufacture of a medicament for treating or preventing ATTR in a subject. The pharmaceutical composition comprises a human anti-TTR antibody or antigen-binding fragment thereof capable of binding to mutated, misfolded, misassembled, and / or aggregated TTR species and / or fragments thereof, and does not substantially recognize physiological TTR species. The pharmaceutical composition further comprises one or more (e.g., 1, 2, or 3) of sucrose, polysorbate 80, and a polar excipient. Preferably, the pharmaceutical composition is a pharmaceutical composition of the present invention.

[0085] In some embodiments, the pharmaceutical composition comprises a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL to about 150 mg / mL (e.g., about 50 mg / mL, about 60 mg / mL, 70 mg / mL, about 80 mg / mL, 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL), a histidine buffer at a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, for use in the manufacture of a medicament for treating or preventing ATTR in a subject. The anti-TTR antibody or antigen-binding fragment thereof can bind to mutated, misfolded, misassembled, and / or aggregated TTR species and / or fragments thereof, and does not substantially recognize physiological TTR species.

[0086] The present invention further relates to a method for obtaining the pharmaceutical composition of the present invention, which comprises formulating the antibody in a buffer as defined above. [Brief explanation of the drawings]

[0087] The accompanying drawings illustrate embodiments of the present disclosure and are included to provide a further understanding of its implementation. [Figure 1] 1 is a graph showing size exclusion chromatography (SEC) % main peak trends for the indicated buffers in the pH buffer study described in Example 1. [Figure 2] 1 is a graph showing capillary isoelectric focusing (cIEF) % main peak trends for the indicated buffers in the pH screening study described in Example 1. [Figure 3] 1 is a graph showing SEC % main peak reduction for six formulations (F) after incubation at 40° C., as described in Example 1. [Figure 4] 1 is a graph showing the % main peak reduction of SEC for the indicated buffers after two weeks of incubation at 40° C., as described in Example 1.

[0088] definition For the avoidance of doubt, it is emphasized that expressions such as "in some embodiments," "in certain embodiments," "in certain examples," "in some examples," "in further embodiments," "in one embodiment," "in a further aspect," "in a first aspect," "in a second aspect," and the like are used and meant to indicate that any of the embodiments described herein should be read in light of the combination of each of the features of those embodiments, and that the present disclosure should treat combinations of features of those embodiments and aspects in the same way as if they were written out as a single embodiment. The same applies to any combination of features of the embodiments and appended claims shown in the Examples, which are also intended to be combined with features from the corresponding embodiments disclosed herein. For consistency and brevity only, the embodiments are characterized by a dependency relationship; however, in practice, each embodiment and combination of features that may be interpreted due to a dependency relationship(s) should be considered literally disclosed, and not as a selection among different alternatives. In this context, those skilled in the art will understand that the embodiments and features disclosed in the Examples are intended to be generalizable to any anti-TTR antibody and equivalents having substantially the same properties.

[0089] As used herein, the term "about" refers to a value that is within ±10%, preferably ±5%, of the recited value. For example, "about 8%" can mean any percentage between 7.2% and 8.8%, preferably any percentage between 7.6% and 8.4%. In another example, "about 2 mL" can mean any volume between 1.8 mL and 1.2 mL (e.g., 1.8 mL, 1.9 mL, 1.95 mL, 2 mL, 2.05 mL, 2.10 mL, 2.15 mL, and 2.2 mL). In the context of antibody amounts referred to herein, e.g., 50 mg / mL or 100 mg / mL, the term "about" refers to concentrations ranging from 45 mg / mL to 50 mg / mL (see "Acceptance Criteria" defined in the Examples), preferably 48 mg / mL to 52 mg / mL (see, e.g., Tables 4, 54, 56, 57, 60, 62, 64, 65, 67, and 68), and concentrations ranging from 90 mg / mL to 113 mg / mL, preferably 96 mg / mL to 113 mg / mL (see, e.g., Tables 15, 20, 28, 34, and 46). Thus, even when the term "about" is not explicitly used, the above concentration ranges still apply, e.g., a reference to 50 mg / mL includes concentrations ranging from 45 mg / mL to 55 mg / mL, and a reference to 100 mg / mL includes concentrations ranging from 90 mg / mL to 113 mg / mL, because these ranges are within experimental variation as shown in the tables above.

[0090] In the context of the present invention, the term "and / or" is understood to mean that all members of the group connected by the term "and / or" are disclosed alternatively to one another and in each case cumulatively with one another in any combination. This means that for the expression "A, B and / or C", the following disclosure is to be understood accordingly: a) A or B or C; or b) (A and B); or c) (A and C); or d) (B and C); or e) (A and B and C).

[0091] The phrases "essentially free of NaCl" and "substantially free of NaCl" in the context of the formulations / pharmaceutical compositions of the present invention mean that the pharmaceutical composition / formulation does not contain NaCl or contains only trace amounts that are considered negligible for the intended use of the product, i.e., NaCl is present at such low levels that it does not affect the performance, stability, safety, or efficacy of the formulation. Furthermore, these phrases also mean that NaCl is not intentionally added, but is present due to the presence of salts of other excipients, such as histidine-HCl. 2+ or Cl - It may also refer to a formulation / pharmaceutical composition that may include any of the above.

[0092] The phrases "essentially free of" and "substantially free of" in the context of the formulations / pharmaceutical compositions of the present invention mean that the pharmaceutical composition / formulation does not contain the referenced substance or contains only trace amounts that are considered negligible for the intended use of the product, i.e., the substance is present at such low levels that it does not affect the performance, stability, safety, or efficacy of the formulation.

[0093] As used herein, the term "binding capacity" refers to a characteristic that corresponds to a quantitative measure of biological activity (e.g., TTR binding). Binding capacity assays (e.g., ELISA assays) can be used to measure the ability of an anti-TTR antibody or antigen-binding fragment thereof of the present disclosure to elicit a specific response in a disease-relevant system (e.g., a subject with ATTR-CM, e.g., WT-ATTR-CM). The activity measured in the assay is a surrogate for the intended biological effect and can be used to assess maintenance of that effect over time (e.g., after storage).

[0094] As used herein, the phrases "capable of binding" and "binds to" refer to the ability of an antibody or antigen-binding fragment thereof to bind, e.g., aggregated TTR, under experimental conditions such as those illustrated in the Examples (e.g., in an ELISA assay). As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent (e.g., an anti-TTR antibody described herein), optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers. Pharmaceutical compositions are formulated for administration to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a particular disease or condition that affects or may affect the subject (e.g., ATTR-CM such as WT-ATTR-CM).

[0095] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, to be contacted with the tissues of a subject, e.g., a mammal (e.g., a human), without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0096] As used herein, the term "between" is inclusive of the endpoints.

[0097] As used herein, room temperature (RT) is defined as between 15 and 25°C according to the European Pharmacopoeia. "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or relative to a given nucleic acid or amino acid sequence B (alternatively, it can be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and Y is the total number of nucleic acids in B. It is understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A. DETAILED DESCRIPTION OF THE INVENTION

[0098] The present disclosure relates to pharmaceutical compositions containing anti-transthyretin (TTR) antibodies or antigen-binding fragments thereof that can be used in the various therapeutic and prophylactic methods described herein. The pharmaceutical compositions contain antibodies or antigen-binding fragments thereof that can bind to mutated, misfolded, misassembled, and / or aggregated TTR species and / or fragments thereof, and that do not substantially recognize physiological TTR species.

[0099] The pharmaceutical compositions contain a combination of particular carriers and excipients that impart surprisingly beneficial properties to the compositions, including the ability to formulate the drug (anti-TTR antibodies and antigen-binding fragments thereof) at high concentrations (e.g., about 150 mg / mL), enhance the stability of the compositions (e.g., extended shelf life, e.g., about 4 weeks, particularly at elevated temperatures (e.g., about 40°C) and 18 months at lower temperatures (e.g., about 5°C)), reduce drug aggregation (e.g., ≦400 particles per mL that are ≧10 μM in size), and improve the viscosity parameters of the compositions (e.g., about 16 cP). The components of the pharmaceutical compositions are described in more detail below.

[0100] Antibodies and antigen-binding fragments thereof The pharmaceutical compositions described herein comprise antibodies or antigen-binding fragments, e.g., human anti-TTR antibodies, that can bind to aggregated human wild-type transthyretin (wtATTR) and preferably do not bind to human native TTR monomers and dimers. Preferably, the antibodies can also bind to mutant TTR aggregates.

[0101] Exemplary heavy chain variable (VH) regions, light chain variable (VL) regions, and complementarity determining regions (CDRs) of the anti-TTR antibodies described herein are shown in Table 1 below. CDR sequences were defined according to the Kabat system (bioinf.org.uk / abs / ). SEQ ID NO: 1 (VH-CDR1) represents residues 31-35 (Kabat numbering) of SEQ ID NO: 7 (VH). SEQ ID NO: 2 (VH-CDR2) represents residues 52-67 (Kabat numbering) of SEQ ID NO: 7 (VH). SEQ ID NO: 3 (VH-CDR3) represents residues 100-109 (Kabat numbering) of SEQ ID NO: 7 (VH).

[0102] SEQ ID NO:4 (VL-CDR1) represents residues 31-35 (Kabat numbering) of SEQ ID NO:8 (VL). SEQ ID NO:5 (VL-CDR2) represents residues 52-67 (Kabat numbering) of SEQ ID NO:8 (VL). SEQ ID NO:6 (VL-CDR3) represents residues 100-109 (Kabat numbering) of SEQ ID NO:8 (VL).

[0103] [Table 1-1]

[0104] [Table 1-2] CDR = complementarity determining region; VH = heavy chain variable region; VL = light chain variable region; HC: heavy chain; LC: light chain ** The N-terminal glutamine is absent and / or modified to pyroglutamic acid (also called N-terminal cyclization), preferably the N-terminal glutamine is modified to pyroglutamic acid

[0105] An anti-TTR antibody or antigen-binding fragment thereof may comprise one or more CDR sequences comprising an amino acid sequence having about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6. Additionally, an anti-TTR antibody or antigen-binding fragment thereof may comprise one or more CDR sequences having an amino acid sequence containing one, two, or three mismatches to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and / or SEQ ID NO: 6. In certain examples, an anti-TTR antibody or antigen-binding fragment thereof comprises six CDR amino acid sequences having 100% sequence identity to the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. An anti-TTR antibody or antigen-binding fragment thereof may have a VH region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO:7. In certain examples, an anti-TTR antibody or antigen-binding fragment thereof has a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7. An anti-TTR antibody or antigen-binding fragment thereof may have a VL region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In a particular example, an anti-TTR antibody or antigen-binding fragment thereof has a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0106] An anti-TTR antibody or antigen-binding fragment thereof may have a VH region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In a particular example, an anti-TTR antibody or antigen-binding fragment thereof has a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 11.

[0107] An anti-TTR antibody or antigen-binding fragment thereof may have a VL region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In a particular example, an anti-TTR antibody or antigen-binding fragment thereof has a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 12.

[0108] Alternatively, the anti-TTR antibody is an anti-TTR antibody described in U.S. Patent No. 10,344,080 or U.S. Patent No. 11,180,545 (each of which is incorporated by reference herein in its entirety). In some embodiments, the anti-TTR antibody is an anti-TTR antibody described in U.S. Patent Application Publication No. 2022-0144928 (which is incorporated by reference herein in its entirety).

[0109] Also within the scope of the present disclosure are anti-TTR antibodies and antigen-binding fragments thereof in which specific amino acids have been substituted, deleted, or added. These modifications do not substantially affect the biological properties of the anti-TTR antibodies, such as binding activity. For example, the antibodies may have amino acid substitutions in the framework regions (FR) to improve antigen binding. In another example, some acceptor framework residues may be replaced with corresponding donor amino acids. The donor framework may be a mature or germline human antibody framework sequence or a consensus sequence. Guidance regarding methods for making phenotypically silent amino acid substitutions is provided, for example, by Bowie et al. (Science, 247:1306-1310, 1990), Cunningham et al. (Science, 244:1081-1085, 1989), Ausubel (ed.) (Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1994), T. Maniatis, E.F. Fritsch, and J. Sambrook (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY, 1989), Pearson (Methods Mol. Biol. 243:307-31, 1994), and Gonnet et al. (Science 256:1443-45, 1992), each of which is incorporated herein by reference.

[0110] The variant antibodies or antigen-binding fragments thereof are functionally active and may have less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% of the number of residues substituted or deleted while retaining essentially the same immunological properties, including, but not limited to, binding to TTR, as described herein, i.e., equivalent antibodies having substantially the same binding properties for TTR as the exemplary antibodies described in U.S. Pat. Nos. 10,344,080(B2) and 11,180,545(B2) (corresponding to International Application WO 2015 / 092077(A1) in which the antibody is designated NI-301.37F1; see also above) comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:7 and a light chain variable region having the amino acid sequence of SEQ ID NO:8. As described above, this antibody (designated antibody NI301A) is characterized in U.S. Patent Nos. 10,344,080 (B2) and 11,180,545 (B2) and Michalon et al., Nat. Commun. 12 (2021), 3142, by (i) high-affinity, selective binding to disease-associated ATTR aggregates, (ii) binding to misfolded / aggregated wild-type and variant TTR associated with sporadic and genetic diseases, respectively, and to ATTR deposits in cardiac tissue obtained at autopsy from ATTR-CM patients, but not to native TTR monomers, and importantly, by its ability to remove ATTR fibrils by macrophage-mediated phagocytosis, as described in WO 2020 / 094883 (A1), see also Michalon et al., (2021), supra.

[0111] Antibodies or antigen-binding fragments thereof can also include variants that exhibit biological activity, e.g., binding of an antigen such as TTR, including, for example, humanized or chimeric antibodies or antigen-binding fragments thereof, antibody analogs, orthologs, homologs, and derivatives. Antibodies can contain one or more analogs of an amino acid (including, for example, unnatural amino acids, amino acids that occur naturally only in unrelated biological systems, modified amino acids from mammalian systems, etc.), antibodies with substituted bonds, and other modifications known in the art.

[0112] In some embodiments, the anti-TTR antibody fragment is selected from the group consisting of bis-Fab, Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments.

[0113] In certain embodiments, the anti-TTR antibody or antigen-binding fragment thereof is a monoclonal antibody (mAb). In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof is an IgG antibody. In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof is a human IgG antibody. In certain embodiments, the human anti-TTR antibody or antigen-binding fragment thereof does not elicit an anti-drug antibody (ADA) response in a human subject. In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof has a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7 and a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0114] In this regard, those skilled in the art recognize that effector function and potency may depend, inter alia, on the IgG class or isotype, and that IgG2 and IgG4 have reduced effector function compared to IgG1 or IgG3. Thus, in one embodiment, the anti-TTR antibodies described herein may be of the IgG1 or IgG3 class or isotype, e.g., IgG1. Of course, in addition to using native IgG immunoglobulins, corresponding effector functions can be engineered. See, e.g., Saunders KO (2019) Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front. Immunol. 10:1296. Doi:10.3389 / fimmu.2019.01296.

[0115] The five major classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. They are distinguished by the type of heavy chain found in the molecule. IgG molecules have heavy chains known as gamma chains. IgM have mu chains. IgA have alpha chains. IgE have epsilon chains, and IgD have delta chains. For a review, see, e.g., Schroeder et al., J. Allergy Clin. Immunol. 125 (2010), S41-S52. Furthermore, different subclasses exist: IgA is further classified into subclasses IgA1 and IgA2, and IgG is further classified into subclasses IgG1, IgG2, IgG3, and IgG4. Furthermore, there are two types of light chains: kappa (κ) and lambda (λ).

[0116] In principle, antibodies used according to the present invention may be of any type of class and subclass and may contain any type of light chain, as long as the antibody binds to misfolded, preferably aggregated, TTR, preferably as shown in the Examples of WO 2015 / 092077 A1 for antibody NI-301.37F1, and its binding specificity for TTR remains essentially unaffected, and as long as no adverse effects occur when the antibody is administered to a subject, which can be determined as described in Example 1. However, preferably, complete IgG antibodies containing constant domains are used. Thus, in one embodiment, the immunoglobulin heavy and / or light chain constant domains present in the antibody used according to the present invention are of the IgG, IgM, IgA, IgD, or IgE type, preferably of the IgG type. In one embodiment, the immunoglobulin heavy and / or light chain constant domains present in the antibodies used according to the invention are of the IgA1, IgA1, IgG1, IgG2, IgG3, or IgG4 subclass, preferably the IgG1, IgG2, IgG3, or IgG4 subclass, most preferably the IgG1 subclass.

[0117] Recombinant expression of fully human IgG1 antibodies with human or mouse constant domains can be performed substantially as described in the Examples of WO 2015 / 092077 A1. Preferably, the antibodies are monoclonal or derived from monoclonal antibodies.

[0118] Not only are there four subclasses of IgG, but human heavy and light chain genes also exhibit extensive structural polymorphism, are closely related, and are inherited as haplotypes. Allotypic variants are immunogenic and can elicit antibody responses as a result of alloimmunization. Therefore, allotype switching may be particularly interesting for providing non-immunogenic antibody therapeutics. Although a wide range of allotypes (polymorphisms) is known, research has focused on serologically defined allotypes. IgG protein allotypes are defined by the expression of unique epitopes recognized by distinct serological reagents. Allotypes expressed in the constant region of the IgG heavy chain are designated Gm (genetic marker) along with the subclass, e.g., G1m1 [or G1m(a)], G3m5 [or G3m(b1)], and the allotype number (or letter), e.g., G1m1 [or G1m(a)], G3m5 [or G3m(b1)]. Human immunoglobulin allotypes are listed in Table 1 of Jefferis and Lefrance, mAbs 1 (2009), 1-7 and in Figure 1A of Irani et al., Molecular Immunology 67 (2015), 171-182, the contents of which are incorporated herein by reference. Thus, in one embodiment, the antibody used in accordance with the present invention is of any one of the following allotypes: G1m1, G1m2, G1m3, G1m17, G2m23, G3m21, G3m28, G3m11, G3m5, G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1, A2m2, A2m3, Em1, Km1, Km2, and Km3, but is preferably of the G1m2, G1m3, or G1m17 allotype, and most preferably of the G1m3 allotype.

[0119] As explained above, antibody NI006 / ALXN2220 is a fully human IgG1m3 allotype antibody, composed of two identical heavy chains of the IgG1 subclass and the IgG1m3 allotype. Also, as mentioned above, since the original human antibody NI-301.37F1 is a κ type, NI006 / ALXN2220 is composed of two identical light chains of the κ subclass. The sequences of the variable heavy chain (VH) and variable light chain (VL) of NI006 / ALXN2220 are shown in SEQ ID NOs: 2 and 6, and the sequences of the corresponding human constant regions are known in the art. For example, each isotype, such as the IgG1m3 isotype, has a unique amino acid sequence of the constant region of its heavy chain. See Jefferis and Lefrance (2009), supra. Thus, in one embodiment, the antibody present in the pharmaceutical formulation of the invention is characterized by two heavy chains, each heavy chain (HC) comprising the amino acid sequence set forth in SEQ ID NO: 9, and two light chains (LC), each light chain (LC) comprising the amino acid sequence set forth in SEQ ID NO: 10. Each heavy chain consists of 450 amino acid residues, and each light chain consists of 214 amino acid residues. The four chains are stabilized by intra- and inter-chain disulfide bonds, and the positions of the disulfide bridges identified by Lys-C and trypsin digestion and subsequent LC-MS (see Example 2) are as follows: LC:C23-LC:C88 LC:C134-LC:C194 LC:C214-HC:C223 HC:C22-HC:C97 HC:C147-HC:C203 HC1:229-HC2:229 and HC1:232-HC2:232 HC:C264-HC:C324 HC:C370-HC:C428. (Amino acid numbering corresponds to the heavy chain sequence set forth in SEQ ID NO: 9)

[0120] Thus, the antibodies present in the pharmaceutical composition of the invention may be characterized as containing at least eight disulfide bridges, preferably at the positions specified above.

[0121] Furthermore, each heavy chain of antibody NI006 / ALXN2220 contains a single N-linked glycosylation site at Asn300. The N-linked glycosylation structure is predominantly a fucosylated, complex, biantennary glycan with zero galactose residues (GOF) (approximately 49%) or one galactose residue (GIF) (approximately 25%). A detailed glycosylation profile is shown in Example 2. Glycosylation plays an important role in the stability, in vivo activity, solubility, serum half-life, and immunogenicity of many therapeutic proteins. N-glycan analysis determines the relative distribution of N-glycans released from glycoproteins and provides insightful information regarding the safety and efficacy of biological therapeutics.

[0122] Thus, in a preferred embodiment, the antibody present in the pharmaceutical composition of the invention is an IgG antibody and has a heavy chain that is N-glycosylated, preferably the N-linked glycosylation site is Asn300, and preferably, when expressed, for example, in CHO cells, the antibody comprises N-linked glycosylation structures that are predominantly glycans with zero galactose residues (GOF) (about 49%) or one galactose residue (G1F) (about 25%). Most preferably, the antibody has a glycosylation profile as shown in Example 3.

[0123] Additionally, one or several amino acids at the amino or carboxy termini of the light and / or heavy chains, e.g., the C-terminal lysine of the heavy chain, if present, may be partially or completely deleted or derivatized in the molecule.

[0124] Thus, in one embodiment, the antibody present in the pharmaceutical composition of the invention has a heavy chain that does not contain a C-terminal lysine. For example, in such an embodiment, the C-terminal lysine contained in SEQ ID NO: 9 is missing. The sequence of such a heavy chain is set forth in SEQ ID NO: 13.

[0125] Additionally or alternatively, the antibody has a heavy chain in which the N-terminal glutamine is derivatized, preferably replaced with pyroglutamic acid. This pyroglutamic acid formation is also referred to as N-terminal cyclization. The sequence of such a heavy chain is set forth in SEQ ID NO: 14 or SEQ ID NO: 15.

[0126] Most preferably, the antibody has a heavy chain that is C-terminally lysine-free, i.e., C-terminally lysine clipped, N-terminally glutamine substituted with pyroglutamic acid, i.e., N-terminally glutaminyl cyclized (see SEQ ID NO: 15), and N-glycosylated.

[0127] The amino acid sequences of the heavy and light chains are shown below:

[0128] [Table 2]

[0129] (SEQ ID NO: 9, NI006 / ALXN2220, heavy chain amino acid sequence, where constant region amino acids are underlined, the C-terminal lysine (K) is optional and / or the N-terminal glutamine (Q) undergoes intramolecular cyclization, resulting in the formation of pyroglutamic acid)

[0130] [Table 3]

[0131] (SEQ ID NO: 10, NI006 / ALXN2220, light chain amino acid sequence, where the constant region amino acids are underlined).

[0132] Furthermore, the theoretical molecular weight of antibody NI006 / ALXN2220 is 144.2 kDa, and the weights determined by mass spectrometry (MS) are 144.2 kDa (deglycosylated) and 147.0-147.6 kDa (intact IgG1), respectively. Thus, in one embodiment, the antibody contained in the pharmaceutical composition of the present invention has a molecular weight of about 150 kDa, preferably about 147 kDa.

[0133] formulation The present disclosure features pharmaceutical compositions containing the above-described anti-TTR antibodies or antigen-binding fragments thereof. Pharmaceutical compositions of the present invention can be formulated as described below. For example, pharmaceutical compositions containing anti-TTR antibodies can be formulated to contain sucrose, polysorbate, preferably polysorbate 80, and / or polar excipients (e.g., histidine). Furthermore, pharmaceutical compositions containing anti-TTR antibodies can be formulated at a desired pH (e.g., pH 5.8) as described herein. Pharmaceutical compositions containing anti-TTR antibodies can further contain a pharmaceutically acceptable excipient or diluent as described herein.

[0134] For example, the pharmaceutical composition may contain a human anti-TTR antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7 and a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:8, and an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9 and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:10, respectively), a histidine buffer having a pH of about 5.8, 6.5% (w / v) sucrose, and 0.03% (w / v) polysorbate 80 in a volume of 2.0 mL.

[0135] In another example, the pharmaceutical composition may comprise a human anti-TTR antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7 and a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:8, and an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9 and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:10, respectively), a histidine buffer having a pH of about 5.8, 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, in a total volume of 2.0 mL.

[0136] As shown in Example 3, about 99% to 100% of the antibodies present in a pharmaceutical formulation have an N-terminal pyroglutamic acid in their heavy chains, and about 96% of the antibodies have a loss of a C-terminal lysine. Thus, in one embodiment, about 99% of the antibodies in a formulation of the invention have heavy chains in which an N-terminal pyroglutamic acid has been modified from an N-terminal glutamine, and / or about 96% of the antibodies have a loss of a C-terminal lysine.

[0137] Thus, the pharmaceutical composition may contain, in a volume of 2.0 mL, a human anti-TTR antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 and a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15, preferably SEQ ID NO: 15, and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10), a histidine buffer having a pH of about 5.8, 6.5% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, respectively.

[0138] In another example, the pharmaceutical composition may contain a human anti-TTR antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 11 and a VL region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 12, 13, 14, or 15, preferably SEQ ID NO: 15, and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10), a histidine buffer having a pH of about 5.8, 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, in a total volume of 2.0 mL.

[0139] In addition, to a small extent, preferably in negligible amounts, some antibody species may be found in the analyzed antibody composition that may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamic acid formation and isomerization. PTMs identified to be present in NI006 / ALXN2220 are mentioned in Example 3. In particular, following the above-mentioned C-terminal lysine clipping and N-terminal cyclization, the antibody may exhibit, for example, methionine (M) oxidation at HC position 255; asparagine (N) deamidation at, for example, HC position 318 and / or HC position 387; asparagine (N) succinimide formation at, for example, HC position 318; and / or amidation of the C-terminal proline (P) following loss of the C-terminal lysine and glycine.

[0140] antibody concentration Any of the anti-TTR antibodies or antigen-binding fragments thereof described herein (e.g., anti-TTR antibodies having a VH region having the amino acid sequence of SEQ ID NO: 7 or 11 and a VL region having the amino acid sequence of SEQ ID NO: 8 or 12, respectively, and anti-TTR antibodies having a heavy chain having the amino acid sequence of SEQ ID NO: 9 or any one of SEQ ID NOs: 13 to 15 and a light chain having the amino acid sequence of SEQ ID NO: 10) can be formulated into the pharmaceutical compositions described herein at a concentration of about 1 mg / mL to about 500 mg / mL.

[0141] The pharmaceutical composition may have a concentration of about 1 mg / mL to about 400 mg / mL (e.g., about 1 mg / mL to about 100 mg / mL, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, or about 100 mg / mL, e.g., about 100 mg / mL to about 200 mg / mL, e.g., about 100 mg / mL, about 110 mg / mL, about 125 mg / mL, about 150 mg / mL, about 175 mg / mL, or about 200 mg / mL, e.g., about The pharmaceutical composition may comprise an anti-TTR antibody or antigen-binding fragment thereof described herein at a concentration of 200 mg / mL to about 300 mg / mL, e.g., about 200 mg / mL, about 210 mg / mL, about 225 mg / mL, about 250 mg / mL, about 275 mg / mL, or about 300 mg / mL, e.g., about 300 mg / mL to about 400 mg / mL, e.g., about 300 mg / mL, about 310 mg / mL, about 325 mg / mL, about 350 mg / mL, about 375 mg / mL, or about 400 mg / mL. The pharmaceutical composition may comprise an anti-TTR antibody or antigen-binding fragment thereof described herein at a concentration of about 1 mg / mL to about 300 mg / mL. The pharmaceutical composition may comprise an anti-TTR antibody or antigen-binding fragment thereof described herein at a concentration of about 1 mg / mL to about 200 mg / mL.The pharmaceutical composition may have a concentration of about 1 mg / mL to about 100 mg / mL (e.g., about 1 mg / mL to about 20 mg / mL, e.g., about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, or about 20 mg / mL, e.g., about 20 mg / mL to about 40 mg / mL, e.g., about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, or about 40 mg / mL, about 40 mg / mL to about 60 mg / mL, e.g., about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, e.g., about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about The pharmaceutical composition may comprise an anti-TTR antibody or antigen-binding fragment thereof described herein at a concentration of about 0 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, or about 60 mg / mL, e.g., about 60 mg / mL to about 80 mg / mL, e.g., about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, or about 80 mg / mL, e.g., about 80 mg / mL to about 100 mg / mL, e.g., about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL. The pharmaceutical composition may comprise an anti-TTR antibody or antigen-binding fragment thereof described herein at a concentration of about 30 mg / mL to about 70 mg / mL. The pharmaceutical composition can comprise about 40 mg / mL to about 60 mg / mL (e.g., about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, or about 60 mg / mL) of an anti-TTR antibody or antigen-binding fragment thereof described herein.

[0142] The pharmaceutical composition may comprise about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL of an anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise about 50 mg / mL of an anti-TTR antibody or antigen-binding fragment thereof described herein.

[0143] sucrose The pharmaceutical composition may also contain, for example, sucrose in an amount of about 6% to about 9%, about 6% to about 7%, or about 7.5% to about 8.5% (w / v) by weight per unit volume (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9% (w / v) sucrose). For example, the pharmaceutical composition may contain about 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.45%, 6.5%, 6.55%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.05%, 8.1%, 8.2%, 8.3%, 8.4%, or 8.5% (w / v) sucrose. In particular, the pharmaceutical composition contains about 6.5% (w / v) sucrose or about 8% (w / v) sucrose.

[0144] Polysorbate The pharmaceutical compositions may also include a polysorbate, such as polysorbate 20 or polysorbate 80, preferably polysorbate 80 (PS80), in an amount of, for example, about 0.001% to about 0.1% (w / v) (e.g., about 0.001%, 0.005%, 0.01%, 0.05%, or 0.1% (w / v) PS(80)). For example, the pharmaceutical composition can contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% (w / v) PS80. In particular, the pharmaceutical composition contains about 0.03% (w / v) PS(80).

[0145] Polar excipients / buffer systems The pharmaceutical composition may also include a polar excipient. The polar excipient may be or include, for example, a sugar, a polyol, or an amino acid. The sugar may be, for example, sucrose, trehalose, fructose, lactose, dextrose, or mannitol. The polyol may be, for example, polyethylene glycol or sorbitol. The amino acid may be, for example, one or more of alanine, arginine, aspartic acid, asparagine, carnitine, citrulline, ornithine, glycine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, and valine. In a preferred embodiment, the polar excipient used as a buffer in the formulation of the present invention is histidine (e.g., L-histidine and / or L-histidine monohydrochloride, or a pharmaceutically acceptable salt thereof). In some embodiments, the polar excipient is L-histidine and / or L-histidine monohydrochloride, or a pharmaceutically acceptable salt thereof. As known to those skilled in the art, polar excipients include buffers, which are well known in the art and include, for example, citrate buffer, phosphate buffer, acetate buffer, histidine buffer, and combinations thereof. Suitable buffers can be selected using, inter alia, the guidelines above. A preferred buffer for use in accordance with the present invention is a histidine buffer. Pharmaceutical compositions can include a polar excipient (buffer) (e.g., histidine) in an amount of, for example, about 1 mM to about 100 mM (e.g., about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM).For example, the pharmaceutical agent may be present in an amount of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 19.5 mM, about 20 mM, about 20.5 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, About 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 3 8mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51m M, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61mM, about 62mM, about 63mM, about 64mM, About 65mM, about 66mM, about 67mM, about 68mM, about 69mM, about 70mM, about 71mM, about 72mM, about 73mM, about 74mM, about 75mM, about 76mM, about 77mM, about 7 The pharmaceutical composition may comprise 8 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, or about 100 mM of a polar excipient (e.g., histidine). In particular, the pharmaceutical composition comprises about 20 mM of a polar excipient (e.g., histidine).

[0146] pH The pharmaceutical composition may have a pH of about 5.0 to about 8.0 (e.g., about 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). For example, the pharmaceutical composition may have a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.75, 5.8, 5.85, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In particular, the pharmaceutical composition has a pH of about 5.8.

[0147] Excipients and Diluents Pharmaceutical compositions may contain pharmaceutically acceptable excipients (e.g., buffers, carriers, stabilizers, or preservatives) or diluents (e.g., saline and aqueous buffer solutions). Pharmaceutically acceptable buffer solutions include, for example, bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0148] volume The pharmaceutical composition may be administered in a volume of about 1 mL to about 100 mL, about 1 mL to about 50 mL, about 5 mL to about 25 mL, about 20 mL to about 25 mL, or about 1 mL to about 10 mL (e.g., about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 5 mL to about 20 mL, about 5 mL to about 15 mL, about 5 mL to about 10 mL, about 10 mL The composition may be provided (e.g., in a vial or other container, as described herein) in a volume of about 1 mL to about 20 mL, about 15 mL to about 20 mL, about 20 mL to about 22.5 mL, about 20 mL to about 25 mL, about 1 mL to about 9 mL, about 1 mL to about 8 mL, about 1 mL to about 7 mL, about 1 mL to about 6 mL, about 1 mL to about 5 mL, about 1 mL to about 4 mL, about 1 mL to about 3 mL, about 1 mL to about 2.25 mL, about 1 mL to about 2 mL, or about 2 mL to about 2.25 mL. For example, the pharmaceutical composition may be about 1 mL to about 2.25 mL (e.g., about 1 mL to about 2.2 mL, about 1 mL to about 2 mL, about 1 mL to about 1.8 mL, about 1 mL to about 1.6 mL, about 1 mL to about 1.4 mL, about 1 mL to about 1.2 mL, about 1.5 mL to about 1.25 mL, about 1.5 mL to about 2 mL, about 1.9 mL to about 1.2 mL, about 2.1 mL to about 2.25 mL) or about 1 mL to about 100 mL (e.g., about The composition may be present in a volume of about 1 mL, about 1.8 mL, about 1.9 mL, about 2 mL, about 2.1 mL, about 2.2 mL to about 2.25 mL, about 2.3 mL, about 2.4 mL, about 2.5 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL.

[0149] In a particular example, the pharmaceutical composition may be present in a volume of about 2.25 mL or about 2 mL. In another example, the pharmaceutical composition may be present in a volume of about 20 mL or about 22.5 mL.

[0150] Additional preparations The pharmaceutical composition may be prepared such that it has not been reconstituted from lyophilized anti-TTR antibody and / or has not been further lyophilized. The pharmaceutical composition may also be prepared such that it is essentially free of sodium chloride and / or essentially free of poloxamer. The pharmaceutical composition may also be prepared as a sterile composition.

[0151] In a most preferred embodiment of the invention, the pharmaceutical composition comprises or consists of 50 mg / mL antibody (antibody NI006 / ALXN2220 characterized above), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), 65 mg / mL or 80 mg / mL sucrose, preferably 80 mg / mL sucrose, 0.3 mg / mL polysorbate 80, and water for infusion / injection, pH 5.8.

[0152] Features The present disclosure features the invention of pharmaceutical compositions having improved characteristics (e.g., stability, solubility, storage, etc.) as described herein (see, e.g., Examples 1 and 2).

[0153] The pharmaceutical composition may be characterized as having a shelf life of 24 months at 2-8°C, preferably protected from light.

[0154] The pharmaceutical composition is characterized by one, two, three, or all four of the following stability criteria (e.g., (a)-(d)) in any combination: (a) a main peak decrease of less than 1% by weight of the antibody under heat stress conditions at 40°C for 4 weeks and / or at about 25°C for 12 weeks, as measured by size exclusion chromatography (SEC)-HPLC analysis; (b) the pharmaceutical composition exhibits an acidic species content of the anti-TTR antibody of less than 42.5% under heat stress conditions at about 40°C for 2 weeks, as measured by capillary isoelectric focusing (cIEF); (c) the pharmaceutical composition (d) the anti-TTR antibody exhibits no substantial change in acidic species content after three cycles of freeze-thaw (from about -70°C to room temperature (e.g., 20-26°C, e.g., 25°C)) as measured by capillary isoelectric focusing (cIEF); and / or (d) the anti-TTR antibody retains at least 80% of its binding ability to TTR protein after 4 weeks of storage at about 40°C and / or at least 70% of its binding ability to TTR protein after 12 weeks of storage at about 25°C, as measured, for example, by ELISA and compared to a control (e.g., no long-term storage).

[0155] For example, the pharmaceutical composition exhibits a main peak ≧50.0%, an acidic peak ≦40.0%, and a basic peak ≦15.0% as measured, for example, by cIEF; a main peak (monomer) ≧95.0% and high molecular weight species (HMWS) ≦5.0% as measured, for example, by size exclusion chromatography (SEC)-HPLC analysis; a pH of 5.8±0.5; an osmolality of ≧240 mOsm / Kg; and an antibody concentration of 50±5.0 mg / mL.

[0156] Additionally, the pharmaceutical composition of the present invention may be characterized by one, two, three, or all four stability criteria (i)-(iv) in any combination: (i) the main peak decline (representing monomer content) during long-term storage at about 40°C for 1 month, or at about 25°C for 6 months, or at about 5°C for 18 months, as measured by SEC-HPLC, is less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2%; (ii) the pharmaceutical composition loses about 40% or less of the anti-TTR antibody or its antigen under heat stress conditions at about 40°C for 2 weeks, or at about 25°C for 3 months, as measured by cIEF. (iii) the pharmaceutical composition exhibits an acidic species content of the anti-TTR antibody or antigen-binding fragment thereof under long-term storage conditions of 12 or 18 months at about 5°C as measured by cIEF of less than 40%, preferably less than 35%; and / or (iv) the anti-TTR antibody or antigen-binding fragment thereof retains at least 80%, preferably at least 90%, of its binding ability to TTR protein after 6 months of storage at about 25°C or after 12 or 18 months of storage at about 5°C compared to a control (e.g., without long-term storage) as measured by ELISA.

[0157] Preferably, the pharmaceutical composition exhibits any one or all of the characteristics set out in Tables 56-69 of the Examples.

[0158] In particular, the pharmaceutical composition has an osmolality of 240 mOsm / Kg or greater and comprises sucrose (e.g., about 6.5% or about 8% (w / v)), and optionally a surfactant (e.g., PS80 in an amount of about 0.03% (w / v)).

[0159] The pharmaceutical composition is stable upon freezing and thawing. As used herein, the term "stable" or "stability" used in the context of the pharmaceutical compositions described herein refers to the maintenance of the physical and functional characteristics of the composition over time. For example, a stable composition can be described as retaining its appearance (e.g., color, opalescence, number of visible particles, and / or number of subvisible particles), pH, antibody concentration, and / or osmolality after extended storage (e.g., days or weeks), after storage at various temperatures (e.g., -70°C, 4°C, 25°C, or 40°C), and / or after one or more freeze-thaw cycles (e.g., 1, 2, 3, 4, 5, or more). As another example, a stable composition can be described as one that retains its function (e.g., anti-TTR binding ability, as described herein) after storage at various temperatures (e.g., -70°C, 4°C, 25°C, or 40°C) and / or after one or more freeze-thaw cycles (e.g., 1, 2, 3, 4, 5, or more).

[0160] The pharmaceutical composition of the present invention has been shown to remain stable for at least 1 month at 40±2°C and 75±5% RH (stressed stability test); at least 6 months at 25±2°C / 60±5% RH (accelerated stability test); and at least 12 to 18 months at 5±3°C (long-term stability test). Furthermore, the extinction coefficient of the pharmaceutical composition is 1.438 (mg / mL). -1 cm -1 The pharmaceutical formulation was shown to be a sterile, colorless to slightly yellow, clear to slightly opalescent solution, essentially free of visible particles, at a pH of 5.8.

[0161] Treatment methods An anti-TTR antibody or antigen-binding fragment thereof (e.g., a VH region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 7 (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 7, e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 7, or 100% sequence identity to SEQ ID NO: 11, e.g., also having the CDR sequences set forth in SEQ ID NOs: 1-3) and an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8 (e.g., at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 8, e.g., Pharmaceutical compositions containing anti-TTR antibodies or antigen-binding fragments thereof comprising a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and a VL region comprising a CDR sequence set forth in SEQ ID NO:13, 14, or 15, preferably SEQ ID NO:15, and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:10, respectively, can be administered to a subject (e.g., a human) to treat, prevent, or control the diseases or disorders described herein. For example, the pharmaceutical compositions described herein can be used in methods for treating or preventing transthyretin-mediated amyloidosis (ATTR). Furthermore, the pharmaceutical compositions described herein can be used in methods for treating or preventing ATTR amyloidosis with cardiomyopathy (ATTR-CM, e.g., WT-ATTR-CM).For example, human anti-TTR antibodies or antigen-binding fragments thereof (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region comprising an amino acid sequence having at least 100% sequence identity to the amino acid sequence of SEQ ID NO:7, or having 100% sequence identity to SEQ ID NO:11, and a VL region comprising 100% sequence identity to the amino acid sequence of SEQ ID NO:8, as well as an anti-TTR antibody or antigen-binding fragment thereof having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and an anti-TTR antibody or antigen-binding fragment thereof having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, Preferably, a pharmaceutical composition comprising a PTM as described herein above, i.e., an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 13, 14, or 15, preferably SEQ ID NO: 15, and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, at a concentration of about 50 mg / mL, in a histidine buffer having a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, can be administered to treat ATTR or ATTR-CM, e.g., WT-ATTR-CM.

[0162] Pharmaceutical compositions containing anti-TTR antibodies can be administered to human subjects to treat, prevent, or control transthyretin-mediated amyloidosis (ATTR), including ATTR amyloidosis with cardiomyopathy (ATTR-CM, e.g., WT-ATTR-CM). The pharmaceutical compositions can be administered in doses ranging from 30 to 60 mg / kg (e.g., 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, or 60 mg / kg) of human anti-TTR antibody or up to 75,000 mg (e.g., 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, 3000mg3000mg, 3100mg, 3200mg, 3300mg, 34 00mg, 3500mg, 3600mg, 3700mg, 3800mg, 3900mg, 4000mg, 4100mg, 4200mg, 4300mg, 4400mg, 4500mg, 46 00mg, 4700mg, 4800mg, 4900mg, 5000mg, 5100mg, 5200mg, 5300mg, 5400mg, 5500mg, 5600mg, 5700mg, 58 The human anti-TTR antibody can be administered by intravenous injection or infusion at a dose providing a therapeutically effective amount of human anti-TTR antibody (e.g., 3000 mg, 5900 mg, 6000 mg, 6100 mg, 6200 mg, 6300 mg, 6400 mg, 6500 mg, 6600 mg, 6700 mg, 6800 mg, 6900 mg, 7000 mg, 7100 mg, 7200 mg, 7300 mg, 7400 mg, or 7500 mg). For example, a 3000 mg dose or a 7500 mg dose, as well as doses in between, can be used to treat a human subject.

[0163] The compositions and methods provided herein can be used to treat subjects with ATTR, ATTR-CM, ATTR polyneuropathy (ATTR-PN), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, meningeal / central nervous system (CNS) amyloidosis, Alzheimer's disease, TTR-related ocular amyloidosis, TTR-related renal amyloidosis, TTR-related hyperthyroxinemia, TTR-related ligament amyloidosis, carpal tunnel syndrome, rotator cuff tear, lumbar spinal stenosis, preeclampsia, or a known pathogenic TTR mutation (e.g., one that causes amyloidosis). The subject can have sporadic WT-ATTR-CM and have a negative genetic test for a TTR mutation.

[0164] The pharmaceutical compositions of the present disclosure can be formulated for administration by various methods known in the art.Administration can be, for example, intravenous or subcutaneous.Intravenous delivery by continuous infusion is one method for administering the pharmaceutical compositions disclosed herein.

[0165] The pharmaceutical composition can be ready to use for administration to a subject in need thereof, preferably by intravenous infusion. The pharmaceutical composition can be diluted with glucose or a polymer thereof prior to injection, preferably the polymer is dextran. The concentration of glucose or a polymer thereof can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w / v).

[0166] In some examples, the pharmaceutical composition is administered in the dosage regimen described in ClinicalTrials.gov ID NCT04360434 and Garcia-Pavia et al. (2023) and European Patent Application Nos. 22 207 651.5 and 23 020 175.8; U.S. Patent Application No. 63 / 383,807; and International Application No. PCT / EP2023 / 081809, filed November 15, 2023, which claims priority to European Patent Application Nos. 22 207 651.5 and 23 020 175.8 and U.S. Patent Application No. 63 / 383,807 (incorporated herein by reference). Thus, in a preferred embodiment, the pharmaceutical compositions of the present invention, particularly the most preferred compositions of NI006 / ALXN2220 referenced above, are administered approximately every 28 days.

[0167] manufactured goods The disclosure also features articles of manufacture (e.g., kits) containing materials useful for treating or preventing transthyretin-mediated amyloidosis (ATTR) in a human subject. The articles of manufacture include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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, 7, 48, 49, 50, or more than 50) containers and labels or package inserts on or associated with the one or more containers. Suitable containers include, for example, bottles (e.g., infusion bottles), vials (e.g., Type I clear glass vials), syringes, IV solution bags, etc. Containers such as Type I clear glass vials or infusion bottles can be 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL in size and can accommodate an overfill of the pharmaceutical composition by about 10% to about 15% (e.g., 10% or 12.5%) by volume. The container may contain a volume of a pharmaceutical composition described herein, such as, for example, 0.5 mL to 10 mL, 2 mL to 2.25 mL, 10 mL to 20 mL, 15 mL to 25 mL, or 20 mL to 22.5 mL, such as a volume of 0.5 mL, 1 mL, 2 mL, 2.25 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 20.5 mL, 21 mL, 21.5 mL, 22 mL, 22.5 mL, 23 mL, 24 mL, or 25 mL. In a preferred embodiment, the pharmaceutical composition, i.e., the pharmaceutical product described herein, is provided as a concentrate for solution for infusion, presented as a sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles, and supplied in a 2 mL (2R) glass vial with an aluminum flip-off cap over a 13 mm rubber stopper. The product is preferably diluted with sterile glucose, a commercially available product, prior to administration, which, in one embodiment, is not provided with the pharmaceutical product.

[0168] The pharmaceutical compositions include, for example, a human anti-TTR antibody or antigen-binding fragment thereof (e.g., an anti-TTR antibody or antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least 100% sequence identity to the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:11 and a VL region comprising 100% sequence identity to the amino acid sequence of SEQ ID NO:8, as well as an anti-TTR antibody or antigen-binding fragment thereof having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and an anti-TTR antibody or antigen-binding fragment thereof having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, preferably a pharmaceutical composition comprising a PTM as described herein above, i.e., SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 ... and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10) at a concentration of about 1 mg / mL to about 150 mg / mL (e.g., about 50 mg / mL, about 60 mg / mL, 70 mg / mL, about 80 mg / mL, 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, or about 150 mg / mL), and may contain, for example, a histidine buffer having a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80.The pharmaceutical composition of the manufactured product is, for example, an anti-TTR antibody or an antigen-binding fragment thereof comprising a VH region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 7 (e.g., having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 11, and having, for example, the CDR sequences described in SEQ ID NOs: 1-3), and a VL region comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8 (e.g., having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO: 8, and having, for example, the CDR sequences described in SEQ ID NOs: 4-6), and an anti-TTR antibody having a heavy chain comprising an amino acid sequence having the amino acid sequence of SEQ ID NO: 9, preferably, a pharmaceutical composition comprising the PTM described above herein, i.e., an anti-TTR antibody having a heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 13, 14, or 15, preferably SEQ ID NO: 15, and a light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 10), in a total amount of 2500 mg to about 7500 mg (e.g., about 2500 mg to about 3000 mg, about 2750 mg to about 3500 mg, about 3000 mg to about 4000 mg, about 3500 mg to about 4500 mg, about 4000 mg to about 5000 mg, about 4500 mg to about 5500 mg, about 5000 mg to about 6000 mg, about 5500 mg to about 6500 mg, about 6000 mg to about 7000 mg, or about 6500 mg to about 7500 mg, e.g., 2500 mg, 2750 mg, 3000 mg, 3250 mg, 3500 mg, 3750 mg, 4000 mg, 4250 mg, 4500 mg, 4750 mg, 5000 mg, 5250 mg, 5500 mg, 5750 mg, 6000 mg, 6250 mg, 6500 mg, 6750 mg, 7000 mg, 7250 mg, or 7500 mg), and may contain together, for example, a histidine buffer having a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80.The pharmaceutical composition of the product contains a total amount of about 2500 mg to about 5000 mg (e.g., 2500 mg to 3000 mg, 2750 mg to about 3500 mg, 3000 mg to about 4000 mg, 3500 mg to 4500 mg, or 4000 mg to 5000 mg, e.g., 2500 mg, 2750 mg, 3000 mg, 3250 mg, 3500 mg, 3750 mg, 4000 mg, 4250 mg, 4500 mg, 4750 mg, or 5000 mg) of an anti-TTR antibody or antigen-binding fragment thereof (e.g., having a VH region having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:7 or SEQ ID NO:11, e.g., having a CDR sequence set forth in SEQ ID NOs:1 to 3), and a CDR sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:8. and an anti-TTR antibody or antigen-binding fragment thereof comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10 (e.g., having at least 85%, 90%, 95%, 97%, 99%, or 100% sequence identity to SEQ ID NO:8, e.g., having the CDR sequences set forth in SEQ ID NOs:4-6), in a total volume of about 2 mL to about 25 mL (e.g., 2 mL, 2.25 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 20.5 mL, 21 mL, 21.5 mL, 22 mL, 22.5 mL, 23 mL, 24 mL, or 25 mL).

[0169] The container can be formed from a variety of materials, such as glass or plastic. The container can hold a pharmaceutical composition described herein that is effective for treating or preventing ATTR, either by itself or in combination with another composition. The container can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-TTR antibody described herein. The label or package insert indicates that the composition is used for treating ATTR.

[0170] The article of manufacture may include at least a first container having a pharmaceutical composition contained therein, the pharmaceutical composition comprising an anti-TTR antibody described herein. Optionally, the article of manufacture may further include a second container having a second therapeutic agent. The article of manufacture in this embodiment of the present disclosure may further include a package insert indicating that the composition can be used to treat ATTR. Alternatively, or in addition, the article of manufacture may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as BWFI, PBS, Ringer's solution, and dextrose solution.

[0171] The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. For example, the article of manufacture (e.g., a kit) may include a second container containing a pharmaceutically acceptable buffer such as phosphate-buffered saline, Ringer's solution, and / or glucose or a polymer thereof, such as dextran (e.g., at a concentration of about 5% (w / v)). The article of manufacture may include other materials, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0172] In a preferred embodiment, the article of manufacture comprises a vial, preferably a clear glass vial sealed with a (gray) rubber stopper and a (blue) aluminum-plastic cover flip-off cap. Preferably, the antibody is present in the vial at a concentration of 50 mg / mL and is provided as a concentrate for infusion solution, present as a sterile, colorless to slightly yellow, clear to slightly opalescent liquid, essentially free of visible particles.

[0173] In a further preferred embodiment, the article of manufacture comprises a dosing syringe in a dosing pump or infusion bag containing the diluted antibody formulation. [Example]

[0174] The following examples of specific modes for carrying out the present disclosure are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0175] Example 1. Formulation development of anti-TTR monoclonal antibodies This example summarizes the results of formulation development of an anti-transthyretin (TTR) monoclonal antibody (mAb) having a heavy chain with the amino acid sequence of SEQ ID NO:9 and a light chain with the amino acid sequence of SEQ ID NO:10, each obtained by recombinant expression in CHO-K1 cells. The physical and chemical stability of the anti-TTR antibody was evaluated to design a stable formulation for clinical development. Experiments were designed to evaluate the effects of excipients on antibody properties under accelerated and stress conditions. Biophysical and analytical methods were used to determine potential chemical and physical changes that occurred in the antibody formulated under these various conditions.

[0176] The goal of the formulation development studies was to identify a stable formulation (e.g., pharmaceutical composition) for the antibody, as determined according to pH buffer studies, solubility studies, excipient screening studies, and surfactant screening studies.

[0177] Analysis method exterior The appearance of all samples, including clarity, color, and visible particles, was examined against a black and white background using a YB-2 light box.

[0178] Caliper-SDS R / NR Caliper-sodium dodecyl sulfate (SDS) reduced (R) / non-reduced (NR) electrophoresis was performed on a PerkinElmer Caliper automated electrophoresis system. Samples were treated with sample buffer, SDS, and N-ethylmaleimide (for non-reduced, i.e., NR) or dithiothreitol (for reduced, i.e., R) at 70°C for 10 minutes. A minimum volume of 42 μL of sample (final antibody concentration 0.045 mg / ml) was then run on a LABCHIP® GXII TOUCH™ at excitation / emission wavelengths of 635 nm and 700 nm. Final results were analyzed using commercially available software: LABCHIP® GX Reviewer.

[0179] cIEF Capillary isoelectric focusing (cIEF) was performed on a PROTEINSIMPLE® cIEF analyzer equipped with an FC-coated cIEF cartridge. 50 μg of each sample was mixed with 90 μl of a master mix consisting of isoelectric point (pI) markers 7.65 / 9.46, PHARMALYTE™ 3-10, PHARMALYTE™ 8-10.5, 1% methylcellulose, and 8 M urea. After mixing, the samples were focused at 1500 V for 1 minute and 3000 V for 8 minutes, respectively. The detection wavelength was set at 280 nm to evaluate the charge variant distribution in different pI ranges.

[0180] DLS Zeta potential was measured by Malvern dynamic light scattering (DLS). Samples were diluted to 5 mg / mL and 1 mL of each sample was placed in the DLS sample cell.

[0181] ELISA binding The assay was an enzyme-linked immunosorbent assay (ELISA) for antibody binding capacity. Appropriate dilutions of samples and reference standards were loaded onto half-area 96-well plates coated with misfolded TTR. After washing, the antibody product was added to the wells. After washing the plate, HRP-conjugated goat anti-human IgG was added to the wells and allowed to interact with the antibody captured during the previous step. After a final washing step, TMB substrate solution (Cat. N301, THERMO SCIENTIFIC™) was loaded into the wells. TMB specifically reacts with peroxide in the presence of peroxidase, producing a colorimetric signal proportional to the amount of antibody bound to the well. Color development was stopped, and the optical density was measured at 450 / 560 nm. Dose-response curves for samples, controls, and reference standards were plotted according to a four-parameter logistic (Auto-Estimate) regression model using SOFTMAX® Pro Software. Individual EC50s were calculated for the samples and reference standards, respectively. The relative binding activity of the samples was calculated using the EC50 of the reference for the samples on the same plate.

[0182] Osmotic pressure Osmolality was measured using an osmometer. Before and after testing, the accuracy of the osmometer method was confirmed using a clinitrol 290 milliosmoles (mOsm) dinitrol reference solution. The sample volume for testing was 20 μL, and only one test was performed for each sample.

[0183] MFI Subvisible particles were monitored by a microfluidic imaging (MFI) system. Approximately 1.5 ml of each sample volume was transferred to an MFI 96-well plate in a biosafety hood for analysis. Results were analyzed by the vendor's software. The amount of subvisible particles at equivalent circular diameters between >2 μm, >10 μm, and >25 μm was reported.

[0184] Particulate matter (high precision (HIAC)) A HACH particle analyzer was utilized to measure sub-visible particle size and count under a laminar flow cabinet. All samples were kept in the cabinet for at least 0.5 hours before testing to avoid the introduction of air bubbles and interference during testing. Each sample was tested four consecutive times with 0.45 mL each. Results were expressed as the average number of particles ≥ 10 μm and ≥ 25 μm per mL (methods were as per USP <788> Particulate matter in injections).

[0185] pH The pH of the samples was measured using a pH meter equipped with a glass electrode, which was calibrated before each use.

[0186] antibody concentration Antibody concentrations were determined by a THERMO™ UV spectrophotometer. The extinction coefficient used in all evaluation studies was 1.390 AU. * mL * mg -1* cm -1 All measurements were repeated twice with 2.5 μL of sample each time, and the average results were reported. For concentrations above 100 mg / mL, samples were diluted to 20 mg / mL before testing.

[0187] SEC-HPLC Size exclusion chromatography (SEC) was performed on an AGILENT® high-performance liquid chromatography (HPLC) system equipped with an SEC column (300 × 7.8 mm, 5 μm). The sampler temperature was set at 5 ± 3 °C, and the column oven temperature was set at 25 ± 3 °C. The mobile phase was 50 mM PB, 300 mM NaCl, pH 6.8 ± 0.1, and the flow rate was set at 1.0 mL / min. Samples were diluted to 10 mg / mL with the mobile phase, and 100 μg of sample was injected. The detection wavelength was set at 280 nm, and the run time was 20 min.

[0188] Turbidity (UV350) Turbidity was measured using a spectrophotometer (SPECTRA MAX™). 150 μL of sample was added to each well of a 96-well plate, and 150 μL of each buffer solution was also added to the corresponding well as a reference. The absorbance of the buffer and sample was then tested at 350 nm. The UV350 value of the antibody was obtained by subtracting the corresponding buffer solution.

[0189] viscosity Viscosity was measured with a Brookfield viscometer using a CP-51 cone and plate geometry. Approximately 0.5 mL of sample was used for each measurement, and a Brookfield viscosity standard (29.14 cP) was used before sample measurement.

[0190] Preformulation Research To identify optimal formulations (e.g., pharmaceutical compositions) of anti-TTR antibodies, pH / buffer screening studies and solubility studies were performed, as detailed below.

[0191] pH / Buffer Screening Studies This study aimed to screen the most suitable pH buffer conditions for anti-TTR antibodies.

[0192] Sample preparation Nine candidate buffers with different pH values ​​were used in this study, and details of these buffers are shown in Table 2. Three buffer systems were used: 20 mM acetate, 20 mM histidine, and 20 mM phosphate buffer (PB), with pH values ​​ranging from 4.5 to 7.5. Drug substance (DS) (formulated in 20 mM histidine, pH 6.0) produced from a 50 L pool was used in this study. The DS was buffer-exchanged into three prepared buffers (20 mM acetate pH 5.0, 20 mM histidine pH 6.0, and 20 mM PB pH 7.0) by dialysis. Six other pH buffer conditions were achieved by adjusting the solution with the corresponding acid or base. The anti-TTR antibody concentration used in this study was 50 mg / mL. Each of the prepared samples was filtered and filled into 2 mL glass vials (1 mL / vial), then stoppered, capped, and immediately labeled.

[0193] [Table 4]

[0194] Study parameters Table 2 shows the conditions evaluated for the pH / buffer screening study. Samples were stored at 40°C for 4 weeks. Samples were collected at appropriate time points and maintained at 2-8°C prior to analysis. Test parameters performed for this study included appearance, pH, antibody concentration, SEC-HPLC, cIEF, Caliper-SDS (non-reduced (NR) and reduced (R)), dissociation constant (kD) values ​​by DLS, and zeta potential, as further detailed below. Selected sample F6 was tested for antigen binding by ELISA at the 4-week (w) time point.

[0195] [Table 5] X = appearance, pH, antibody concentration (A280), SEC-HPLC, cIEF, Caliper-SDS (NR&R); Y = kD value (DLS), zeta potential; Z = Elisa binding capacity (only for F6); w = week

[0196] Results of the pH / Buffer Screening Study Appearance, pH, and protein concentration Summary data on appearance, pH and antibody concentration in the pH screening study are listed in Table 4.

[0197] For samples in acetate or histidine buffer (F1–F6), the samples exhibited a pale yellow, slightly opalescent color with visible particles at TO and maintained a similar appearance after 4 weeks of incubation at 40°C. Of these six samples, F2 (20 mM acetate pH 5.0) exhibited a small number of particles at TO but later showed no visible particles after incubation at 40°C. For F6 (20 mM histidine, pH 6.5), a small number of particles were observed at TO, and a large number of particles were observed at the 4-week time point.

[0198] In these six samples, the appearance of the samples was closely related to the pH value: as the pH increased, more particles were observed and the samples became more cloudy in appearance.

[0199] For the phosphate buffer samples (F7–F9), a milky white liquid containing a large number of particles was observed from TO, and the appearance remained even after 4 weeks of incubation at 40°C.

[0200] [Table 6] LY = pale yellow; SO = slightly milky white; O = milky white; P = particles; LP = large amount of particles; w = week

[0201] SEC-HPLC Table 4 shows a data summary of the SEC-HPLC test results in the pH screening study.

[0202] At time 0, the % main peak for samples in acetate or histidine buffers ranged from 97.2% to 97.9%, while the % main peak for samples in PB buffer was below 96.0%. After 4 weeks at 40°C, the histidine buffers (F4–F6) showed the least decrease in monomer purity (0.7–0.8%) of all buffer systems tested, while PB buffer showed the most substantial decrease in monomer percentage (up to 3.9%). This is also shown in Figure 1.

[0203] An increase in the high molecular weight (HMW) peak and an increase in the low molecular weight (LMW) peak were observed in all nine buffers at 40°C after 4 weeks, with the smallest increase in the histidine buffer and the largest increase in the PB buffer.

[0204] [Table 7] ND = not determined; HMW = high molecular weight; LMW = low molecular weight; w = week; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography Table 6 shows a data summary of the cIEF test results in the pH / buffer screening study.

[0205] All candidates showed a decrease in the percentage of the main peak, as well as an increase in the acidic peak and a decrease in the basic peak after 4 weeks of storage at 40°C. Among all candidates, the histidine buffer had the smallest % main peak decrease, ranging from 19.1% to 21.6%, while the PB buffer had the greatest decrease, with a maximum decrease of 48.1% of cIEF % main peak. The trend of cIEF % main peak change is shown in Figure 2.

[0206] [Table 8] cIEF = capillary isoelectric focusing; w = week

[0207] Caliper-SDS-(R&NR) Table 7 summarizes the data for Caliper-SDS-(R&NR) in the pH buffer screening study. For Caliper-SDS Reduced, all candidates showed a decrease in % heavy chain (HC) + light chain (LC), with the histidine buffer candidate showing the least decrease in % HC + LC (maximum decrease of 1.2%) after 4 weeks of incubation at 40°C. However, the acetate buffer candidate's % HC + LC decreased slightly more (to 1.7%), while the PB buffer candidate's % HC + LC decreased substantially after 4 weeks, to a maximum of 4.7%.

[0208] A similar trend was observed in the Caliper-SDS non-reduced results. A decrease in % main peak was observed in all samples after 4 weeks, with histidine buffer showing the smallest decrease of 2.9-3.6%. Acetate buffer's % main peak decreased from 3.0% to 6.0%, while PB buffer again showed the most substantial change in % main peak, decreasing to 13.1%.

[0209] [Table 9] SDS = sodium dodecyl sulfate; R = reduced; NR = non-reduced; HC: heavy chain; LC: light chain; w = week

[0210] ELISA binding ability After 4 weeks of incubation at 40°C, the sample (20 mM histidine, pH 6.0) was sent for an ELISA binding antigen assay. The results showed that the binding antigen of this sample was 105%, indicating that there was no substantial change in the potency of the anti-TTR antibody after 4 weeks of incubation at 40°C when formulated in a histidine buffer at pH 6.0.

[0211] DLS (kD and zeta potential) For all pH buffer candidates, k DThe values ​​were measured by high-throughput DLS. The zeta potentials of all nine samples were measured by DLS, as well as the sample with added NaCl, to determine whether the addition of NaCl could improve antibody stability. The results are summarized in Table 8.

[0212] [Table 10] K D = dissociation constant; PB = phosphate buffer solution

[0213] Regarding kD values, samples in acetate or histidine buffers showed positive values ​​(12.7 mL / g to 36.7 mL / g at 25°C and 14.0 mL / g to 33.0 mL / g at 40°C), while PB buffer showed negative values. This indicated that the interaction between antibodies in acetate or histidine buffers was repulsive, thus limiting aggregation, while antibodies in phosphate buffer showed more attractive interactions, which could possibly lead to more aggregation.

[0214] Regarding zeta potential, samples in acetate or histidine buffers showed larger values ​​compared to samples in phosphate buffer, indicating that the antibody was more stable in these two buffers than in phosphate buffer.

[0215] The zeta potential value in 20 mM histidine pH 6.0 was 12.67 mV, which decreased to 1.80 mV when 200 mM NaCl was added to the buffer, suggesting that the addition of NaCl impaired the colloidal stability of the anti-TTR antibody.

[0216] overview Slightly opalescent particles were observed in all pH buffers. At higher pHs (e.g., phosphate buffer, pH 6.5, 7.0, and 7.5), large amounts of particles and a cloudy solution were observed. After 4 weeks of incubation at 40°C, no substantial changes in appearance, pH value, or antibody concentration were observed.

[0217] In acetate or histidine buffers, the kD and zeta potential values ​​were higher than in phosphate buffers, indicating that the colloidal stability of the antibody was better in acetate or histidine buffers. Addition of NaCl to the buffer decreased the zeta potential of the antibody in 20 mM histidine buffer, pH 6.0, suggesting that NaCl did not improve the colloidal stability of the protein.

[0218] Based on the SEC-HPLC results, the least % main peak loss of all samples was observed for the antibody in histidine buffer (especially pH 5.5 and 6.0) after 4 weeks of storage at 40° C. The antibody in acetate buffer showed a slightly greater % main peak loss, while the antibody in phosphate buffer showed the greatest % main peak loss on SEC-HPLC.

[0219] The smallest decrease in % main peak of cIEF after 4 weeks of incubation at 40°C was detected for antibodies formulated in histidine buffer (pH 5.5 and 6.0), while the largest decrease was observed for antibodies in phosphate buffer.

[0220] Minimal decreases in %HC+LC for Caliper reduced antibody were observed for antibody in acetate buffer (pH 5.0 and 5.5) and histidine (pH 5.5, 6.0, and 6.5) after incubation at 40° C., while substantial decreases in %HC+LC were observed for antibody in acetate buffer (pH 4.5) or phosphate buffer. For Caliper non-reduced, acetate buffer (pH 5.5) and histidine buffer (pH 5.5 and 6.0) show the least decrease in %IgG purity after 40° C. stress.

[0221] Overall, the pH 5.5-6.0 range of histidine was identified as providing relatively more stable conditions for the antibody, with histidine pH 5.8 buffer providing favorable stability.

[0222] Solubility Test The antibody was formulated at a relatively high concentration (150 mg / mL) in 20 mM histidine buffer, pH 5.8, with and without NaCl, and in phosphate-buffered saline (PBS) to study antibody solubility. Antibody stability was studied at 2-8°C and 25°C.

[0223] Sample preparation Three formulations were used for solubility testing, the details of which are listed in Table 9. The three formulations were 20 mM histidine pH 5.8, 20 mM histidine pH 5.8 with NaCl, and PBS. The starting material (e.g., 50 L pool DS) was buffer-exchanged into these buffers and concentrated to >150 mg / mL. All formulation samples were finally filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter, filled into 2 ml glass vials (1 ml / vial), stoppered, and sealed in a biosafety hood.

[0224] [Table 11] PBS = phosphate-buffered saline

[0225] Study parameters Table 10 shows the conditions evaluated for the solubility study. Samples were stored at 25°C or 5°C for 2 days (D). Samples were collected at each time point and kept at 2-8°C before analysis. Test parameters included appearance, antibody concentration, turbidity, and viscosity.

[0226] [Table 12] X = appearance, protein concentration, turbidity (UV350); Y = viscosity

[0227] Solubility study results Appearance and antibody concentration The appearance, turbidity, and antibody concentration for the solubility test are reported in Table 11. Sample (F1) in 20 mM histidine buffer pH 5.8 remained slightly yellow and slightly milky throughout the study and was free of particles. Sample (F2) in 20 mM histidine buffer pH 5.8 containing 155 mM NaCl was slightly yellow, slightly milky, and free of particles at time zero (T0), but became milky after 48 hours of incubation at either 5°C or 25°C. Antibody in PBS buffer (F3) was slightly yellow, milky, and free of particles throughout the study. A large amount of air bubbles was present, and it was very difficult to remove these bubbles in these highly viscous samples.

[0228] No substantial changes in turbidity or antibody concentration were observed for all samples during the study. Variations in turbidity data may be due to the presence of large amounts of air bubbles, and variations in antibody concentration were due to dilution variations.

[0229] [Table 13] SY = slightly yellow; SO = slightly milky white; O = milky white; FP = particle-free; H = time;

[0230] viscosity Samples were diluted to 150 mg / mL for viscosity measurements. Viscosity data are shown in Table 12. All samples exhibited shear-thinning behavior, with 20 mM histidine pH 5.8 containing NaCl having the lowest viscosity (16 cP), while 20 mM histidine pH 5.8 (F1) and PBS buffer (F3) had the highest viscosity (approximately 60 cP).

[0231] [Table 14] cP = centipoise

[0232] overview In solubility studies, three different pH buffer types were investigated at antibody concentrations above 150 mg / mL. Results showed that no substantial changes in antibody concentration or turbidity were observed over 48 hours at 2-8°C and 25°C, indicating that the anti-TTR antibody was stable short-term at a concentration of 150 mg / mL. The 20 mM histidine pH 5.8 buffer exhibited the preferred appearance of the three formulations investigated: slightly yellow, slightly opalescent, and particle-free.

[0233] The viscosity of the sample was high at an antibody concentration of 150 mg / mL.

[0234] These results demonstrate that the anti-TTR antibody has good stability in 20 mM histidine, pH 5.8 buffer.

[0235] Excipient screening studies: Stability at 40°C and during freeze-thaw Sample preparation Six formulations were evaluated in the excipient screen, as listed in Table 13. The starting material (e.g., 50 L pool DS) was formulated in either 20 mM histidine, pH 5.8, or 20 mM acetate-histidine buffer, pH 5.8. Stock solutions were prepared containing 40% weight (w / v) sucrose, 44.2% (w / v) trehalose 2H2O, 40% (w / v) sorbitol, 40% (w / v) l-arginine HCl, and 10% (w / v) polysorbate 80 (PS80) per unit volume. The amounts of DS and excipient stock solutions were calculated and combined based on the formulation recipe. All formulation samples were finally filtered through a 0.22 μm PVDF filter, filled into 2 mL glass vials (1 mL / vial), stoppered, and sealed in a biosafety hood. An antibody concentration of 100 mg / mL was used in this study.

[0236] [Table 15] PS80 = Polysorbate 80

[0237] Study parameters Table 14 shows the detailed design of the excipient screening study. Samples were stored at 40°C for 4 weeks or subjected to 3 or 5 freeze-thaw cycles. Samples were collected at each time point and kept at 2-8°C before analysis. Test parameters performed in this study included appearance, pH, antibody concentration, osmolality, SEC-HPLC, cIEF, Caliper-SDS (reduced and non-reduced), and MFI.

[0238] [Table 16] Test items: X = appearance, pH, antibody concentration, SEC-HPLC, Caliper-SDS (non-reduced and reduced), cIEF, Y = MFI; Z = osmolality, k D DLS);RT=room temperature

[0239] Excipient screening 40℃ stability study results Appearance, pH, antibody concentration and osmolality The appearance, pH, antibody concentration, and osmolality results from the 40°C stability study are shown in Table 15. For samples without PS80 in the formulation (F1-F5), the samples were slightly yellow and slightly milky white during the 4-week study. Samples at TO were particle-free, while samples incubated at 40°C contained particles.

[0240] For the sample containing PS80 in the formulation (F6), the sample remained slightly yellow and slightly opalescent throughout the study and was free of particles, indicating that the addition of PS80 enhanced the stability of the anti-TTR antibody during storage at 40°C.

[0241] When incubated at 40°C for 4 weeks, no substantial changes in pH value or antibody concentration were observed.

[0242] The osmolality of all samples containing excipients ranged from 339 to 423 mOsm / kg.

[0243] [Table 17]

[0244] SEC-HPLC Table 16 shows the data summary of the SEC-HPLC test results in the excipient screening 40°C study. When stored at 40°C for 4 weeks, the main peak percentage for all samples except F4 decreased by about 3.3%, with no substantial difference observed among these five samples. For F4 (arginine HCl as excipient), a slightly more substantial decrease in the % main peak can be observed (3.8%) after 4 weeks of storage at 40°C.

[0245] [Table 18] HMW = high molecular weight; MLW = low molecular weight; w = week; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography

[0246] cIEF Table 17 shows a summary of the cIEF test results in the excipient screening 40° C. study.

[0247] When stored at 40° C. for 4 weeks, the main peak percentage for all samples decreased by approximately 20% compared to the other formulations, with F5 showing a slightly greater decrease (25.8%) in the main peak percentage. A substantial increase in the percentage of acidic peaks and a slight decrease in the percentage of basic peaks was also observed after 4 weeks of incubation at 40° C.

[0248] [Table 19]

[0249] Non-reducing Caliper-SDS Table 18 shows a data summary of the Caliper-SDS non-reduced test results for the excipient screening 40° C. study. At time zero (T0), the purity of all formulations was 97.1%, with a decrease of approximately 3.5% after 4 weeks of storage at 40° C. No substantial differences were observed among the six formulations.

[0250] [Table 20]

[0251] Reduced Caliper-SDS Table 19 shows the data summary of the reduced Caliper-SDS results in the excipient screening 40° C. study. At time zero (T0), the purity of all formulations was approximately 99.0%, with a decrease of approximately 1.6% after 4 weeks of storage at 40° C. No substantial differences were observed among the six formulations.

[0252] [Table 21] HC = heavy chain; LC = light chain; w = week

[0253] Overview of excipient screening and 40°C stability studies In the sample without PS80, a large amount of bubbles and particles were observed. The sample containing 0.02% PS80 contained no visible particles, suggesting that PS80 confers stability to the composition by reducing aggregation of the anti-TTR antibody. All samples were observed to be slightly yellow and slightly opalescent, and no substantial changes were observed in each formulation after 4 weeks of incubation at 40°C.

[0254] No substantial changes in pH or concentration were observed after 4 weeks of incubation at 40° C. The osmolality of all six samples was greater than isotonic.

[0255] A decrease in SEC purity, cIEF main peak, and Caliper R and NR purity was observed for all six formulations, with F4 showing the greatest decrease in SEC purity and F5 showing the greatest decrease in cIEF main peak percentage.

[0256] No substantial difference was observed between F1 and F6, indicating that the addition of PS80 improved the appearance of the samples without compromising the purity of SEC, cIEF, and Caliper R and NR.

[0257] In summary, formulations containing sucrose or sorbitol performed favorably among all formulations based on 40°C stability studies. Furthermore, the addition of PS80 to the 8% sucrose formulation (F6) resulted in no visible particles after incubation at 40°C, indicating that the addition of PS80 enhanced the stability of the anti-TTR antibody during storage at 40°C.

[0258] Excipient study results: Freeze-thaw study In this excipient study, the stability of all six formulations under 3 and 5 cycles of freeze-thaw was evaluated, and the results are shown below.

[0259] Appearance, pH, antibody concentration and osmolality The appearance, pH, and antibody concentration in this freeze-thaw test are shown in Table 20. For samples without PS80 in the formulation (F1-F5), the samples were slightly yellow and slightly milky white after 3 / 5 cycles of freeze-thaw testing. The TO sample did not contain particles, while the freeze-thawed sample had particles.

[0260] For the sample with PS80 in the formulation (F6), the sample remained slightly yellow, slightly opalescent, and free of particles throughout the study, indicating that the addition of PS80 enhanced the stability of the anti-TTR antibody during freeze-thaw cycles.

[0261] No substantial changes in pH value or antibody concentration were observed after up to five freeze-thaw cycles.

[0262] [Table 22] SY: slightly yellow, SO: slightly milky white, P: particles, FP: no particles

[0263] SEC-HPLC Table 21 shows a summary of the SEC-HPLC test results in the excipient screening freeze-thaw study. After ≦5 cycles of freeze-thaw, no substantial changes were observed in SEC-HPLC Main. For -F4 (arginine HCl as excipient), a slight decrease in % Main peak (0.9%) could be observed after 5 cycles of freeze-thaw.

[0264] [Table 23]

[0265] cIEF Table 22 shows a summary of the cIEF test results in the excipient screening study.

[0266] After five freeze-thaw cycles, a 2.5% or 1.5% decrease in the cIEF main peak percentage was observed in formulation 3 (sorbitol as excipient) and formulation 5 (acetate-histidine buffer, sucrose as excipient), respectively. No substantial changes were observed in the other formulations after up to five freeze-thaw cycles.

[0267] [Table 24]

[0268] Non-reducing Caliper-SDS Table 23 shows the data summary of the non-reducing Caliper-SDS test results in the excipient screening freeze-thaw study. At time zero (T0), the purity of all formulations was 97.1%, and no substantial change in purity was observed after five freeze-thaw cycles.

[0269] [Table 25]

[0270] Reduced Caliper-SDS Table 24 shows the data summary of the reduced Caliper-SDS test results in the excipient screening study. At time zero (T0), the purity of all formulations was approximately 99.0%, with no substantial decrease after five freeze-thaw cycles.

[0271] [Table 26] HC = heavy chain; LC = light chain

[0272] Macroscopic particles (MFI) Subvisible particles measured by MFI in the excipient screening study are shown in Table 25. For formulations without PS80 (F1-F5), numerous subvisible particles were detected starting at time zero (T0), whereas the number was much lower for the sample containing PS80 (F6). The addition of PS80 substantially reduced the number of subvisible particles in the samples. No substantial changes were observed for all formulations before and after five freeze-thaw cycles.

[0273] [Table 27] MFI = Microfluidic Imaging; ECD = Equivalent Circular Diameter; AR = Aspect Ratio

[0274] Overview of freeze-thaw research While a large number of bubbles and particles were difficult to identify in the sample without PS80, the sample containing 0.02% PS80 contained no visible particles, suggesting that PS80 confers stability to the composition by reducing aggregation of the anti-TTR antibody. All samples were observed to be slightly yellow and slightly opalescent, and no substantial changes were observed for each formulation after five freeze-thaw cycles. While numerous subvisible particles were detected in the formulation without PS80, a substantial reduction in subvisible particles was observed when PS80 was added to the formulation.

[0275] No substantial changes in pH or concentration were observed after five freeze-thaw cycles.

[0276] For F4 (arginine HCl as excipient), a 0.9% decrease in SEC main peak percentage was observed, while for the other five formulations, substantial changes in SEC main peak percentage were observed. For cIEF, F3 (sorbitol as excipient) or F5 (acetate-histidine as buffer) showed a slight decrease in main peak percentage, but for the other formulations, no substantial change in cIEF % main peak was observed after five freeze-thaw cycles. Comparing all excipients, the formulation containing sucrose had the best antibody stability in 20 mM histidine buffer.

[0277] After five freeze-thaw cycles, no substantial changes in Caliper NR and R were observed for all samples.

[0278] No substantial difference was observed between F1 and F6, indicating that the addition of PS80 improved the appearance and sub-visible particles of the samples without compromising the purity of SEC, cIEF, and Caliper R and NR.

[0279] In summary, the formulation containing sucrose and PS80 performed best among all formulations. For example, the addition of PS80 to the 8% sucrose formulation (F6) did not result in visible particles after one or more freeze-thaw cycles, indicating that the addition of PS80 enhanced the stability of the anti-TTR antibody during storage at 40°C.

[0280] Overview of excipient screening studies Summarizing the 40°C stability and freeze-thaw studies, it was observed that the formulation containing sucrose provided the most stability of the antibody, as evidenced by the lowest reduction in SEC, cIEF, and Caliper purity compared to the other formulations. The addition of PS80 improved appearance and reduced sub-visible particles without substantially any adverse effect on antibody stability.

[0281] 20 mM histidine buffer, 8% or 6.5% (w / v) sucrose, pH 5.8 was chosen as the final buffer and excipient system. The surfactant (PS80) concentration will be evaluated in the next study.

[0282] Surfactant screening research The objectives of this study were to evaluate the effect of PS80 concentration on antibody stability and to evaluate the effect of sucrose concentration on antibody stability under different stress conditions (25°C, agitation, 40°C, and freeze-thaw).

[0283] Sample preparation Six formulations were evaluated in the surfactant screen, as listed in Table 26. Starting material (e.g., 50 L of pooled DS) was buffer-exchanged into 20 mM histidine, pH 5.8. A stock solution of 60% (w / v) sucrose, 10% (w / w) PS80 was prepared. The amounts of DS, excipient stock solution, and surfactant stock solution were calculated, weighed, and mixed based on the formulation formula. All formulation samples were finally filtered through a 0.22 μm PVDF filter, filled into glass vials (2 mL / vial), stoppered, and sealed in a biosafety hood. Note that 2R vials were used for all formulations, except for the freeze-thaw samples, which were filled into 6R samples, because 2 mL in the 2R bottle resulted in vial breakage during freeze-thaw.

[0284] [Table 28] PS80 = Polysorbate 80

[0285] Study parameters Table 27 shows the sampling and testing plan for the surfactant screening study. Samples were also evaluated with agitation at 200 rpm at 25°C for 3 or 7 days (D), or without agitation at 25°C for 3 or 7 days (D). For freeze-thaw testing, samples were completely frozen at -70°C and completely thawed at room temperature for each cycle, with 3 or 5 cycles evaluated. Samples were also maintained at 40°C for up to 2 weeks. Samples were collected in a timely manner and maintained at 2-8°C before analysis. Test parameters performed for this study included appearance, pH, antibody concentration, osmolality, SEC-HPLC, Caliper-SDS (reduced and non-reduced), cIEF, and subvisible particles (HIAC).

[0286] [Table 29] X = appearance, pH, antibody concentration, SEC-HPLC, non-reduced and reduced Caliper-SDS, cIEF, HIAC; Y = osmolality; D = days

[0287] result Appearance, pH, osmolality and antibody concentration Table 28 summarizes the appearance, pH, osmolality, and antibody concentration data for this study. After 7 days of stirring at 200 rpm, all samples remained slightly yellow, slightly milky, and free of particles.

[0288] The pH and antibody concentrations of F1 to F6 remained stable throughout the study, with pH values ​​ranging from 5.8 to 5.9 and antibody concentrations around 100 mg / mL.

[0289] The osmolality of F1 to F3 (8% sucrose) was approximately 340, and the osmolality of F4 to F6 (6.5% sucrose) was approximately 280. Therefore, F1 to F3 and F4 to F6 meet the European Pharmacopoeia limit (Ph.Eur. limit) of >240 mOsm / kg.

[0290] [Table 30] SY: slightly yellow, SO: slightly milky white; D = days

[0291] SEC-HPLC The SEC-HPLC results of the stirring study in surfactant screening are reported in Table 29. After 7 days of stirring, the main peak percentages of all test samples remained stable, and no substantial differences were observed among these six samples.

[0292] [Table 31] D=days; HMW=high molecular weight;LMW=low molecular weight

[0293] cIEF The cIEF results of the surfactant screening agitation study are reported in Table 30. After 7 days of agitation at 200 rpm, no substantial change in the cIEF main peak percentage was observed in all six formulations.

[0294] [Table 32]

[0295] Non-reducing Caliper-SDS The results for non-reduced Caliper-SDS are reported in Table 31. The purity percentage of non-reduced Caliper-SDS for all test samples remained stable, with no substantial differences observed between these samples during the study.

[0296] [Table 33] D=number of days

[0297] Reduced Caliper-SDS The results of the reduced Caliper-SDS surfactant screen agitation study are reported in Table 32. The reduced Caliper-SDS purity percentages of all test samples remained stable, with no substantial differences observed between these samples during the study.

[0298] [Table 34] HC = heavy chain; LC = light chain; D = days

[0299] Particles invisible to the naked eye (HIAC) Table 33 shows the data summary of the sub-visible particles (HIAC) test results for the agitation studies in surfactant screening. After 7 days of agitation, no substantial changes were observed in the sub-visible particles in the six formulations.

[0300] [Table 35] D=number of days

[0301] Overview of Mixing Research The surfactant screening test evaluated the stability of the formulations using agitation for up to 7 days. After stress testing, no substantial differences were observed among the six different formulations.

[0302] Stability at 25°C Appearance, pH, and antibody concentration Table 34 summarizes the appearance, pH, and antibody concentration data for this study. All samples were slightly yellow, slightly opalescent, and free of particles, with no substantial changes observed after 7 days of incubation at 25°C.

[0303] The pH and antibody concentrations of F1 to F6 remained stable throughout the study, with pH values ​​ranging from 5.8 to 5.9 and antibody concentrations around 100 mg / mL.

[0304] [Table 36] D=number of days

[0305] SEC-HPLC The SEC-HPLC results of the surfactant screening 25°C study are reported in Table 35. After 7 days of incubation at 25°C, the main peak percentages of all test samples remained stable and no substantial differences were observed between these samples.

[0306] [Table 37] HMW = high molecular weight; LMW = low molecular weight; w = weeks; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography; D = days

[0307] cIEF The cIEF results of the surfactant screening 25°C study are reported in Table 36. After 7 days of incubation at 25°C, no substantial change in the cIEF main peak percentage was observed for all six formulations.

[0308] [Table 38] cIEF = capillary isoelectric focusing; D = days

[0309] Non-reducing Caliper-SDS The non-reduced Caliper-SDS results of the surfactant screening 25°C study are reported in Table 37. The non-reduced Caliper-SDS purity percentages of all test samples remained stable, with no substantial differences observed between these samples during the study.

[0310] [Table 39] SDS = sodium dodecyl sulfate; D = days

[0311] Reduced Caliper-SDS The reduced Caliper-SDS results are reported in Table 38. The reduced Caliper-SDS purity percentage for all test samples remained stable, with no substantial differences observed between these samples during the study.

[0312] [Table 40] SDS = sodium dodecyl sulfate; D = days; HC = heavy chain; LC = light chain

[0313] Invisible particles (HIAC) Table 39 shows the data summary of the sub-visible particles (HIAC) test results for the 25° C. studies in surfactant screening. After 7 days of incubation at 25° C., no substantial changes in sub-visible particles were observed in the six formulations.

[0314] [Table 41] HIAC=high precision; D=days

[0315] Overview of the 25°C Study Results showed that after incubation at 25°C for up to 7 days, no substantial changes were observed in appearance, pH, antibody concentration, SEC-HPLC % main peak, cIEF % main peak, or subvisible particles (HIAC) for all six formulations.

[0316] Freezing and thawing research In surfactant screening studies, freeze-thaw studies of up to five cycles were used to assess the stability of the formulations.

[0317] Appearance, pH, and antibody concentration Table 40 summarizes the appearance, pH, and antibody concentration data for the freeze-thaw study. After five freeze-thaw cycles, all samples were slightly yellow, slightly opalescent, and free of particles.

[0318] The pH and antibody concentrations of F1 to F6 remained stable throughout the study, with pH values ​​ranging from 5.8 to 5.9 and antibody concentrations around 100 mg / mL.

[0319] [Table 42] SY = slightly yellow; SO = slightly milky white; O = milky white; FP = particle free; D = days

[0320] SEC-HPLC The SEC-HPLC results are reported in Table 41. After five freeze-thaw cycles, the main peak percentages of all test samples remained stable, and no substantial differences were observed between these samples.

[0321] [Table 43] HMW = high molecular weight; MLW = low molecular weight; w = weeks; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography; D = days

[0322] cIEF The cIEF results are shown in Table 42. After five freeze-thaw cycles, no substantial change in the cIEF main peak percentage was observed in all six formulations.

[0323] [Table 44] cIEF = capillary isoelectric focusing; C = cycle(s)

[0324] Non-reducing Caliper-SDS The results for non-reduced Caliper-SDS are reported in Table 43. The purity percentage of non-reduced Caliper-SDS for all test samples remained stable, with no substantial differences observed between these samples during the study.

[0325] [Table 45] SDS = sodium dodecyl sulfate

[0326] Reduced Caliper-SDS The results for reduced Caliper-SDS are reported in Table 44. The purity percentage of reduced Caliper-SDS for all test samples remained stable, with no substantial differences observed between these samples during the study.

[0327] [Table 46] SDS = sodium dodecyl sulfate; HC = heavy chain; LC = light chain

[0328] Invisible particles (HIAC) Table 45 shows the data summary of the sub-visible particles (HIAC) test results for the freeze-thaw studies in surfactant screening. After five cycles of freeze-thaw, no substantial changes in sub-visible particles were observed in the six formulations.

[0329] [Table 47] * Insufficient sample volume for HIAC study due to vial breakage during freeze-thaw.

[0330] Overview of freeze-thaw research Results showed that no substantial changes were observed in appearance, pH, antibody concentration, SEC-HPLC % main peak, cIEF % main peak, or subvisible particles (HIAC) for all six formulations after up to five freeze-thaw cycles.

[0331] 40℃ Research A two-week incubation at 40°C was used in a surfactant screening study to evaluate the stability of these six formulations.

[0332] Appearance, pH, and antibody concentration Table 46 shows the summary data for appearance, pH, and antibody concentration for the 40° C. study. After two weeks of incubation at 40° C., all samples remained slightly yellow, slightly milky, and free of particles.

[0333] The pH and antibody concentrations of F1 to F6 remained stable throughout the study, with pH values ​​ranging from 5.8 to 5.9 and antibody concentrations around 100 mg / mL.

[0334] [Table 48] SY = slightly yellow; SO = slightly milky white; O = milky white; FP = particle-free; w = week

[0335] SEC-HPLC The SEC-HPLC results of the surfactant screening 40°C study are reported in Table 47. After two weeks of incubation at 40°C, a decrease in the percentage of main peak was observed in all six samples, which is shown in Figure 3. Figure 4 shows the different decreases in % main peak among these six formulations, and as the PS80 concentration increases, a more substantial decrease in SEC % main peak can be observed.

[0336] [Table 49] HMW = high molecular weight; MLW = low molecular weight; w = week; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography

[0337] cIEF The cIEF results of the 40°C study of surfactant screening are reported in Table 48. After 2 weeks of incubation at 40°C, a substantial % main peak reduction (approximately 12%) was observed in all formulations, with no apparent differences observed among the six formulations.

[0338] [Table 50] cIEF = capillary isoelectric focusing; w = week

[0339] Non-reducing Caliper-SDS The non-reduced Caliper-SDS results for the surfactant screening 40°C study are reported in Table 49. Non-reduced CE-SDS purity decreased by approximately 1.8%, with no substantial difference observed between these samples during the study.

[0340] [Table 51] SDS = sodium dodecyl sulfate; w = week

[0341] Reduced Caliper-SDS The reduced Caliper-SDS results are reported in Table 50. No substantial changes in reduced Caliper-SDS were observed among the samples during the study.

[0342] [Table 52] SDS = sodium dodecyl sulfate; HC = heavy chain; LC = light chain

[0343] Particles invisible to the naked eye (HIAC) Table 51 shows the data summary of the sub-visible particles (HIAC) test results for the 40°C study in surfactant screening. After two weeks of incubation at 40°C, no substantial changes in sub-visible particles were observed in the six formulations.

[0344] [Table 53]

[0345] Overview of the 40°C Study The results show that no substantial changes in appearance, pH, antibody concentration, caliper reduction, or subvisible particles (HIAC) were observed for all six formulations after incubation at 40°C for up to two weeks. A substantial decrease in SEC-HPLC % main peak and cIEF % main peak can be observed after 40°C incubation. Increasing the PS80 concentration from 0.02% to 0.06% resulted in a decrease in SEC % main peak after two weeks of incubation at 40°C.

[0346] overview No substantial differences were observed in appearance, pH, antibody concentration, subvisible particles, SEC purity, cIEF purity, or Caliper-SDS results in the agitation study, 25°C study, and freeze-thaw study.

[0347] In the 40°C stress study, a decrease in the % main peak in SEC and the % main peak in cIEF was observed. The SEC purity of the samples containing PS80 decreased with increasing PS80, so a lower concentration of PS80 is recommended. A range of 0.02% and 0.04% PS80 was studied, so a concentration of 0.03% PS80 is recommended for the formulation.

[0348] Overall, the target formulation for anti-TTR antibodies was 20 mM histidine buffer at pH 5.8, 8% (w / v) sucrose, and 0.03% (w / v) PS80.

[0349] Example 2. Stability study of anti-TTR monoclonal antibody formulations As outlined in detail above in Example 1, formulation development of NI006 / ALXN2220 included studies designed to select buffer systems and excipients to stabilize the protein. The formulation was developed to prevent product loss and minimize loss of purity and biological activity in response to stresses encountered during manufacturing, storage, shipping, and handling.

[0350] A pH buffer screening study was performed to determine the optimal buffer system for the pharmaceutical formulation. A 20 mM histidine buffer at pH 5.8 was selected as the final buffer system.

[0351] Different types of excipients were evaluated through excipient studies, including disaccharides (such as sucrose and trehalose), amino acids (such as L-arginine HCl), polyhydric alcohols (such as sorbitol), and surfactants (such as polysorbate 80). Samples were incubated at 40°C for up to 4 weeks. Thermal stability, formation of insoluble aggregates, and purity were monitored. Sucrose and polysorbate 80 were selected as the optimal excipients for the NI006 / ALXN2220 formulation because they were shown to minimize loss in SEC, cIEF, and caliper purity, thus preserving product purity.

[0352] In the excipient concentration screening study, three different polysorbate 80 concentrations (0.02%, 0.04%, and 0.06% (w / v)) and two sucrose concentrations (6.5% and 8% (w / v)) were finally tested. Three stress conditions were used for screening: agitation, heat, and freeze-thaw. In the agitation study, samples were placed at 25°C with or without agitation at 200 rpm for up to 7 days. In the heat study, samples were placed at 40°C for up to 2 weeks. In the freeze-thaw study, samples were frozen and thawed for up to five cycles. Appearance, pH, protein concentration, number of subvisible particles, and purity were evaluated.

[0353] A 0.03% (w / v) polysorbate 80 concentration was selected as the surfactant strength because it effectively suppressed subvisible particle formation and maintained high SEC purity. A comparative study was conducted to compare two formulations with 6.5% or 8% (w / v) sucrose concentrations. No substantial differences were observed between the 6.5% and 8% (w / v) sucrose formulations in DSC (differential scanning calorimetry), appearance, pH, protein concentration, SEC, cIEF, CE-SDS (non-reduced and reduced), subvisible particles, and potency after 1 month of incubation at 40°C or 3 months at 25°C.

[0354] The final formulation developed was 20 mM histidine buffer, 8% (w / v) sucrose, 0.03% (w / v) polysorbate 80, and a target concentration of 50 mg / mL of NI006 / ALXN2220 in pH 5.8. The excipients were selected according to their stabilizing effect on the drug product. L-histidine and L-histidine monohydrochloride, at a concentration of 20 mM, stabilize the pH in the liquid state. Sucrose at a concentration of 8% (w / v) alters osmotic pressure to be isotonic, stabilizes the NI006 / ALXN2220 protein against aggregate formation in the liquid state, and serves as a cryoprotectant during freezing and thawing. Polysorbate 80 at a concentration of 0.03% (w / v) was selected to stabilize the NI006 / ALXN2220 protein against surface-induced protein denaturation or aggregation in the liquid state.

[0355] Manufacturing Process Development The drug product manufacturing process consists of melting, pooling, and blending of drug substance, sterile filtration, aseptic filling, stoppering, capping, visual inspection, and bulk packaging. Sterile filtration was selected as the method to obtain the sterile drug product and was performed by using two in-line sterile filters (0.22 μm, PVDF). A water bubble point test was performed on the filter before and after sterile filtration to confirm the filter's integrity. The drug product's compatibility with contacting components on the filling line, the effects of shear stress caused by the peristaltic pump, and stability under light exposure were evaluated to mitigate potential adverse effects on product quality attributes during manufacturing. A nonclinical lot (Lot 201901004) and three clinical lots (Lot 201903038, Lot 201904050, and Lot 20200801) have been completed. The nonclinical and clinical lots used the same fill volume, container closure system, unit operation sequence, and storage conditions. No significant changes were made to the drug manufacturing process between the non-clinical and clinical lots. Minor differences are noted below. A 2L scale was used for non-clinical lots, and a 14L scale was used for clinical lots. The formulation used for non-clinical lot 201901004 was 50 mg / mL in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8. The formulation used for clinical lots 201903038 and 201904050 was 50 mg / mL in 20 mM histidine buffer, 6.5% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8. The formulation used for clinical lot 20200801 was 50 mg / mL in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8. 5L glass bottles were used for pooling and mixing non-clinical lots of drug substance, and 50L mixing bags were used for pooling and mixing clinical lots of drug substance.

[0356] Melting, pooling and blending of bulk API Frozen drug substance stored in 2 L PETG bottles is thawed at room temperature (18-24°C) in a light-shielded room. After complete thawing, the drug substance is pooled into a 50 L mixing bag and stirred at an appropriate speed to observe transfer without foaming. The mixing time is controlled at 15-20 minutes. Samples are taken for pH, protein concentration, osmolality, and bioburden prior to sterile filtration.

[0357] sterile filtration The bulk drug substance is sterile filtered through two series-connected 0.22 μm sterile filters via a peristaltic pump in a Grade A environment into sterile 20 L disposable bags. Filter integrity tests are performed on both filters before and after sterile filtration.

[0358] aseptic filling Aseptic filling is performed in a RABS unit, which completely encapsulates the filler and provides a Grade A environment. The RABS unit separates the operator from the sterile interior. All filling components are autoclaved and aseptically assembled. Sterile, depyrogenated 2 mL (2R) glass vials are filled to a target volume of 2.25 mL. Fill weight checks are performed periodically during the filling process to ensure the fill weight is between 2.233 and 2.442 g / vial.

[0359] Closing The filled vials are automatically stoppered with 13mm rubber stoppers in a RABS unit. The stoppers are steam sterilized at 122°C for 30 minutes.

[0360] Capping The stoppered vials are transferred to a capping machine via a conveyor belt under Grade A laminar flow protection. The stoppered vials are capped with 13 mm plastic aluminum flip-off caps. The caps are steam sterilized at 122°C for 30 minutes.

[0361] Visual inspection A manual 100% visual inspection of the capped vials is performed by manufacturing personnel followed by a statistically based AQL (Acceptable Quality Level) inspection by Quality Assurance. Release and stability samples are taken after the visual inspection.

[0362] Bulk Packaging and Storage The filled drug vials are then bulk packaged and labeled. The bulk packaged drug vials are stored at 2-8°C.

[0363] Container closure system The drug product container closure system consists of 2 mL (2R) Type I glass vials sealed with 13 mm rubber stoppers and 13 mm flip-off aluminum caps. Components were selected for their resistance to sterilization and depyrogenation processes, as well as for non-reactive contact surfaces for optimal protein compatibility. Compatibility of the container closure system with the drug product is evaluated using accelerated and long-term stability studies as described below.

[0364] The integrity of the container closure system was demonstrated by a dye penetration test. Pharmaceutical vials were immersed in a colored dye and held under vacuum before depressurization to examine dye penetration into the vial. The container and closure system achieved 100% airtightness. Container Closure Integrity Testing (CCIT) is performed annually in the stability program using a non-destructive vacuum decay method. Product contact materials, glass vials, and rubber stoppers meet USP and Ph.Eur. requirements and are suitable for parenteral use. Each lot of vials and closures is verified in accordance with a Certificate of Conformance provided by the supplier.

[0365] Analysis method color Color is measured in accordance with Ph.Eur.2.2.2. The chromatic aberration method is used for color measurement.

[0366] transparency The transparency is measured in accordance with Ph.Eur.2.2.1. The light scattering method is used to measure the transparency.

[0367] pH USP <791> and Ph.Eur.2.2.3. pH is measured by potentiometry.

[0368] Osmotic pressure USP <785> and Ph.Eur.2.2.35. Osmolality is determined indirectly by measuring the depression of the freezing point of the solution.

[0369] iCIEF Whole-column imaging capillary isoelectric focusing (iCIEF) is an identity and purity analysis method used to separate proteins according to their isoelectric point (pI) and monitor the percentage of charge variant species in a protein sample. pI is an intrinsic property of a particular protein molecule and is the pH at which the protein molecule has no net charge. Under an external electric field, charge variants migrate along a continuous pH gradient formed by ampholytes and stop at the point where the pH equals their pI. At that pI, the protein has no net charge and is not attracted by any of the electrodes. Therefore, different monoclonal antibody species with different pI values ​​are separated and focused at different positions. The pI values ​​and relative abundance of resolved peaks can be identified and quantified using chromatography software.

[0370] To meet the acceptance criterion of "matching the profile of the reference standard," the electropherogram should show a peak profile comparable to that of the reference standard. Furthermore, the difference in pI values ​​of the main peak between the test sample and the reference standard (mean) should be 0.2 or less.

[0371] SEC-HPLC Size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC) is a purity analysis method that separates proteins based on their size. The stationary phase consists of inert particles packed into a dense three-dimensional matrix. The particles have small pores that allow only species below a certain size to enter. Larger molecules are too large to enter the pores and simply pass through them. Therefore, larger molecules flow through the column faster than smaller molecules. The smaller the molecule, the longer its retention time. After separation, the relative percentages of high molecular weight (HMW), monomeric, and low molecular weight (LMW) species are quantified by UV detection.

[0372] CE-SDS (reduced type) Reduced capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a purity analysis method that separates proteins based on their electrophoretic mobility, with smaller proteins migrating faster than larger proteins. In this method, the test sample is denatured by heating in the presence of SDS. The sample is reduced by adding the reducing agent beta-mercaptoethanol (BME) to the sample solution. Separation is performed through an uncoated capillary, and the protein sample is detected using a photodiode array (PDA) detector at 220 nm. Results are reported as percent purity.

[0373] CE-SDS (non-reducing type) CE-SDS (non-reduced) is a purity analysis method that separates proteins based on their electrophoretic mobility; smaller proteins migrate faster than larger ones. In this method, the test sample is denatured by heating in the presence of SDS. To prevent sulfhydryls from binding to other sulfhydryls, the alkylating reagent N-ethylmaleimide (NEM) is added to the sample solution. Separation is performed through an uncoated capillary, and the protein sample is detected at 220 nm using a PDA detector. Results are reported as percent purity.

[0374] Bioburden Bioburden testing is USP <61> and Ph.Eur.2.6.12. A 10 mL sample of the drug substance is filtered through the sterile surface of a 0.45 μm membrane. The filter membrane is then transferred onto a soybean-casein digest agar culture plate for determination of the total aerobic microbial count (TAMC). Another filter membrane used to filter the 10 mL sample of the drug substance is transferred onto a Sabouraud dextrose agar culture plate for determination of the total yeast and mold count (TYMC).

[0375] endotoxin Bacterial endotoxin testing is performed by kinetic turbidimetric assay and conforms to USP <85> and Ph.Eur.2.6.14. Endotoxins produced by Gram-negative bacteria are detected using a lysate of horseshoe crab amebocytes that coagulates with endotoxins. Endotoxin concentration can be calculated by establishing a correlation between the time required for the reaction mixture to reach a given absorbance or the rate of turbidity development.

[0376] ELISA (binding assay) The binding capacity of the NI006 / ALXN2220 antibody was assessed using an ELISA method. Appropriate dilutions of samples, controls, and reference standards were loaded into half-area 96-well plates coated with misfolded TTR (the antigen for NI006 / ALXN2220). After washing the plate, horseradish peroxidase (HRP)-conjugated goat anti-human IgG was added to the wells, allowing it to interact with the bound NI006 / ALXN2220 antibody captured by misfolded TTR. After a final wash step, TMB substrate solution was loaded into the wells. TMB specifically reacts with peroxide in the presence of peroxidase, generating a colorimetric signal proportional to the amount of NI006 / ALXN2220 protein bound to the well. Color development was stopped, and the optical density was measured at 450 nm (minus 560 nm for wavelength correction).

[0377] The dose-response curves of the samples and reference standards are plotted according to a four-parameter logistic (automatic estimation) regression model using SoftMax Pro GxP software. Individual EC50 values ​​are calculated for the samples and reference standards. The relative binding activity of the samples is calculated using the following formula: Relative binding activity of sample (%) = (EC50 of reference standard / EC50 of sample) x 100%

[0378] Protein concentration Proteins in solution absorb ultraviolet light at a wavelength of 280 nm due to the presence of aromatic amino acids in the protein molecule. According to the Beer-Lambert law, the absorbance (A) of a protein solution at a fixed wavelength is related to the protein concentration (C), the cell path length (l), and the protein's extinction coefficient (ε) as follows: A = Clε. Unlike traditional UV-Vis methods that rely on a single absolute absorbance value, gradient spectroscopy uses cross-sectional data (absorbance vs. path length) to determine a gradient value for quantifying sample concentration using the gradient spectroscopy formula (slope = εC), derived from the Beer-Lambert law.

[0379] Cell-based assays THP-1 is a human monocytic cell line. NI006 / ALXN2220 is an antibody against misfolded TTR. The biological activity of NI006 / ALXN2220 is to stimulate THP-1 cells to produce IL-8 by binding mis-TTR in cell culture. Briefly, approximately 2 x 104 THP-1 cells in assay medium are seeded per well in a 96-well cell culture plate at 100 µL / well. Then, a mixture of serial dilutions of NI006 / ALXN2220 mAb standard (final concentrations: 2000-0.039 µg / mL) and misfolded TTR (final concentration: 10 µg / mL) is added to duplicate wells at 100 µL / well. After 20-24 hours of incubation at 37 °C with 5% CO2, IL-8 production is measured using a human IL-8 ELISA kit.

[0380] suitability study The compatibility of NI006 / ALXN2220 with clinical materials was evaluated. The compatibility with the following clinical administration settings was evaluated: Bags and infusion lines under PVC settings - IV bags, IV lines, and filters are constructed from PVC material Bags and infusion lines in a non-PVC configuration - IV bags, IV lines, and filters are constructed from non-PVC materials Syringe under PVC setting - Perfusion line and filter are made of PVC material Syringe in non-PVC configuration - Perfusion line and filter are constructed of non-PVC material

[0381] Three concentrations (1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL) were tested at 2-8°C for 24 hours followed by 6 hours at 25°C (30 hours total). Saline and glucose were used as diluents for the 1.0 mg / mL and 20.0 mg / mL concentrations. The results are shown in Tables 52 and 53.

[0382] [Table 54] Cl: colorless; SY: slightly yellow; C: clear; FoP: no visible particles; SO: slightly opalescent 1 The concentrations are the same as the NI006 / ALXN2220 drug concentrations.

[0383] [Table 55] 1 The concentrations are the same as the NI006 / ALXN2220 drug concentrations. 2 The variability in results is due to assay variability. All results from in-use compatibility testing are within the acceptance criteria. The results are not considered to be safety relevant given that within the short usage timeframe, the most likely incompatibilities with plastic materials are adsorption (addressed by protein concentration) and visible particle formation (addressed by compendial methods).

[0384] Visible particles were observed in the saline group, indicating that NI006 / ALXN2220 is more unstable in saline. Data show that when glucose was used as a diluent, no substantial changes were observed in appearance, protein concentration, subvisible particles, SEC-HPLC, or ELISA binding assays. NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL is stable for 24 hours at 2-8°C, followed by 6 hours at 25°C (30 hours total). NI006 / ALXN2220 is compatible with the clinically used materials evaluated.

[0385] A concentration of 0.15 mg / mL, 10 times lower than the lowest dose concentration in clinical trials, was also investigated. Variations were observed in protein concentration and ELISA tests. Variations in ELISA tests may be caused by variations in protein concentration, which may be caused by protein adsorption to contact materials. Consequently, it was decided to use 5% glucose as a diluent for clinical use.

[0386] In Table 54 below, batch analysis data is exemplary listed for non-clinical lot 201901004 and clinical lot 201903038.

[0387] [Table 56] 1 No acceptance criteria were established for non-clinical lots and data are reported for informational purposes only. 2 This result is the average of 6 injections from the retest. The original result is 92.2% for (LC+HC). The underlying cause of the original result was not identified.

[0388] The reference standard used was a composition containing 50 mg / mL of antibody formulated in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) PS80 (pH 5.8) and stored in 100 μl filled vials at −70±10° C. Additional testing was performed to qualify the reference standard, and the results are shown in Table 55.

[0389] [Table 57]

[0390] Calibration of protein concentration and potency was performed against a reference standard, the potency of which is assigned a value of 100% relative potency.

[0391] Stability testing Non-clinical lot 201901004 and clinical lot 201903038 were subjected to stability testing. Non-clinical lot (201901004) has 1-month stressed stability data, 6-month accelerated stability data, and 18-month long-term stability data. Clinical lot (201903038) has 1-month stressed stability data, 6-month accelerated stability data, and 12-month long-term stability data. Under stress conditions, both clinical lot (201903038) and non-clinical lot (201901004) showed a significant decrease in the iCIEF main peak (%) and a trend toward an increase in the acidic peak (%), but no significant changes were observed in other purity assays and ELISA binding assays.

[0392] The drug's shelf life is currently set at 24 months at 5±3°C, protected from light. The available in-use stability and compatibility data above indicate that the ready-to-use solution for injection is stable for up to 24 hours at 2-8°C, and then, after dilution with 5% glucose solution, at 25°C for 6 hours. From a microbiological standpoint, the in-use solution should be used immediately. If not used immediately, the in-use shelf life is set at 4 hours at room temperature or 24 hours at 2-8°C.

[0393] Tables 56-59 summarize the available stress data for non-clinical lot 201901004 and clinical lot 201903038.

[0394] [Table 58]

[0395] [Table 59]

[0396] [Table 60]

[0397] [Table 61]

[0398] Tables 60-63 summarize the available accelerated data for non-clinical lot 201901004 and clinical lot 201903038.

[0399] [Table 62]

[0400] [Table 63]

[0401] [Table 64]

[0402] [Table 65]

[0403] Tables 64-69 summarize the available long-term data for non-clinical lot 201901004 and clinical lot 201903038.

[0404] [Table 66]

[0405] [Table 67]

[0406] [Table 68]

[0407] [Table 69]

[0408] [Table 70]

[0409] [Table 71]

[0410] As can be deduced from the stressed, accelerated, and long-term stability data shown in Tables 55-69, the tested pharmaceutical formulations are long-term stable. For example, the formulations remain liquid and free of visible particles, the pH remains constant, and the monomer content measured by SEC-HPLC does not fall below 96%, meaning that the content of HMWS and LMWS remains below 4% under all test conditions. Furthermore, the amount of acidic species, as measured by iCIEF, does not exceed 40% during the long-term stability test, and ELISA binding assays showed that the antibody maintains its binding capacity (does not fall below 70% of the reference standard under all tested conditions, and does not even fall below 95% during the long-term stability test).

[0411] Example 3. Characterization of mature NI006 / ALXN2220 The antibody NI006 / ALXN2220 was produced in the CHO-K1 cell line ATCC No. CCL61 and obtained from cell culture after cultivation in a large-scale production bioreactor. The amino acid sequences of the mature heavy chain (HC) and light chain (LC) of NI006 / ALXN2220 are set forth in SEQ ID NOs: 9 and 10, with the modifications noted below. The total number of amino acids, the number of amino acids in the heavy chain, and the number of amino acids in the light chain of the IgG antibody are 1328, 450, and 214, respectively.

[0412] Further characterization of antibody NI006 / ALXN2222 was primarily performed by standard procedures, such as mass spectrometry. For example, post-translational modifications of NI006 / ALXN2222 were identified using liquid chromatography with tandem mass spectrometry (LC-MS / MS) analysis of fragments of NI006 / ALXN2222 obtained from sequential digestion with Lys-C and trypsin, as well as free sulfhydryl analysis. Characterization of antibody-based therapeutics by LC-MS analysis is a standard procedure and can be performed by those skilled in the art. See, for example, Robotham and Kelly, Approaches to the Purification, Analysis, and Characterization of Antibody-Based Therapeutics (2020), 1-33.

[0413] N-glycan profiling was performed by using PNGase F to release N-glycans, followed by labeling with 2-AB, followed by HILIC (hydrophilic interaction chromatography) separation and fluorescence detection (FLD) using a UPLC system. Individual N-glycan and unknown peaks were quantified by their peak area percentage relative to the total peak area.

[0414] The results are shown below. The molecular weight of antibody NI006 / ALXN2220, as determined by standard mass spectrometry, is approximately 147.1 kDa for the intact IgG1 and 144.2 kDa for the deglycosylated variant.

[0415] The monoclonal antibody NI006 / ALXN2220 is an IgG1 subclass antibody composed of two heavy chains of the IgG1 subclass and two light chains of the kappa subclass. The four chains are stabilized by multiple disulfide bonds. In particular, at least the following disulfide bridges are present in NI006 / ALXN2220, as determined by standard procedures, i.e., Lys-C and trypsin digestion and subsequent LC-MS: LC:C23-LC:C88 LC:C134-LC:C194 LC:C214-HC:C223 HC:C22-HC:C97 HC:C147-HC:C203 HC1:229-HC2:229 and HC1:232-HC2:232 HC:C264-HC:C324 HC:C370-HC:C428 (The corresponding amino acid sequences of HC and LC are shown in SEQ ID NOs: 9 and 10.)

[0416] NI006 / ALXN2220 is a glycoprotein, and the constant region of each heavy chain contains one N-linked glycan site at residue N300. During glycosylation profile determination, the major N-glycan types were shown to be G0F (approximately 49.0%) and G1F (approximately 25.4%). More specifically, the following glycosylation profile (sugar type, location of glycosylation site, etc.) has been determined for NI006 / ALXN2220:

[0417] [Table 72] Note: 1. Glycan nomenclature follows the order HexNac-hexose-fucose-NeuAc-NeuGc, e.g., 23000 is HexNac(2)-hexose(3)-fucose(0)-NeuAc(0)-NeuGc(0). 2. G1Fa and G1Fb are isomers and are classified as G1F. G1F is calculated as the sum of G1Fa and G1Fb using the original unrounded numbers.

[0418] Additionally, N-terminal glutamine modified as pyroglutamic acid (abundance in sample: 99.9%) and C-terminal lysine clipping of the heavy chain (abundance in sample: 95.8%) were identified as the major post-translational modifications. Additionally, minor percentage modifications such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation were experimentally determined as shown in Table 71.

[0419] [Table 73] Note: 1. HC refers to the heavy chain and LC refers to the light chain. 2. The peptide sequences in underlined font were identified as the sites of PTM. 3. * indicates the N-terminal related peptide of the heavy chain, and # indicates the C-terminal related peptide of the heavy chain. 4. / indicates that no PTM was reported. 5. pE(Q) refers to an N-terminal glutamine modified as pyroglutamic acid. 6.-K refers to loss of the C-terminal lysine. 7.-KG amidation (P) refers to the amidation of the C-terminal proline after loss of the C-terminal lysine and glycine.

[0420] In summary, N-linked glycosylation of the heavy chain, modification of the N-terminal glutamine to N-terminal pyroglutamic acid, and C-terminal lysine clipping of the heavy chain are the major post-translational modifications of NI006 / ALXN2220.

[0421] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0422] While the invention has been described in relation to particular embodiments thereof, it will be understood that it is capable of further modifications, and this application is generally intended to cover any variations, uses, or adaptations of the invention which follow principles and come within known or customary practice in the art to which the invention pertains and which may apply to the essential features described above, including such departures from the invention within the scope of the claims.

Claims

1. A pharmaceutical composition comprising a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof capable of binding to aggregated TTR species and substantially not recognizing physiological TTR species, the pharmaceutical composition comprising one or more of sucrose, polysorbate 80, and a polar excipient.

2. 10. The pharmaceutical composition of claim 1, which is an aqueous formulation.

3. 3. The pharmaceutical composition of claim 1, wherein the antibody binds to a mutated or wild-type aggregated FAP receptor species.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody does not bind to a monomer or dimer of human native TTR.

5. 5. The pharmaceutical composition of claim 1, wherein the antibody binds to a TTR epitope comprising or consisting of the amino acid sequence EEEFVEGIY (SEQ ID NO: 49), GELHGLTTIEE (SEQ ID NO: 50), or WEPFA (SEQ ID NO: 51).

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the pharmaceutical composition comprises about 6% to about 9% weight / unit volume (w / v) of sucrose.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the pharmaceutical composition comprises about 6% to about 7% (w / v) sucrose.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pharmaceutical composition comprises about 6.5% (w / v) sucrose.

9. The pharmaceutical composition of any one of claims 1 to 6, wherein the pharmaceutical composition comprises about 7.5% to about 8.5% (w / v) sucrose.

10. 10. The pharmaceutical composition of any one of claims 1 to 6 and 9, wherein the pharmaceutical composition comprises about 8% (w / v) sucrose.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutical composition comprises about 0.001% to about 0.1% (w / v) polysorbate 80.

12. The pharmaceutical composition of any one of claims 1 to 10, wherein the pharmaceutical composition comprises about 0.01% to about 0.05% (w / v) polysorbate 80.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, wherein the pharmaceutical composition comprises about 0.03% (w / v) polysorbate 80.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the pharmaceutical composition has a pH of about 5.3 to about 6.

3.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the pharmaceutical composition has a pH of about 5.8 to about 5.

9.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein the pharmaceutical composition has a pH of about 5.

8.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the pharmaceutical composition has a pH of 5.8±0.

5.

18. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the pharmaceutical composition has a pH of 5.8±0.

1.

19. 19. The pharmaceutical composition of any one of claims 1 to 18, wherein the polar excipient comprises histidine.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein the polar excipient comprises about 1 mM to about 100 mM histidine.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the polar excipient comprises about 20 mM histidine.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the histidine comprises L-histidine and / or L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof.

23. 23. The pharmaceutical composition of any one of claims 1 to 22, wherein the anti-FTP antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region having three complementarity-determining regions (CDRs) set forth in SEQ ID NOS: 1-3 and a light chain variable (VL) region having three CDRs set forth in SEQ ID NOS: 4-6.

24. 24. The pharmaceutical composition of claim 23, wherein the VH region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, and the VL region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

8.

25. 25. The pharmaceutical composition of claim 23 or 24, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 7 and the VL region comprises the amino acid sequence of SEQ ID NO:

8.

26. 25. The pharmaceutical composition of claim 23 or 24, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 11 and the VL region comprises the amino acid sequence of SEQ ID NO:

12.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the antibody comprises a human Ig constant region.

28. The pharmaceutical composition of any one of claims 1 to 27, wherein the antibody is a human IgG.

29. The pharmaceutical composition according to any one of claims 1 to 28, wherein the antibody is of the human IgG1 type.

30. The pharmaceutical composition of any one of claims 1 to 28, wherein the antibody is of the human IgG1m3 allotype.

31. The pharmaceutical composition of any one of claims 27 to 30, wherein the antibody comprises a kappa (κ) light chain.

32. The pharmaceutical composition of any one of claims 1 to 31, wherein the antibody is NI006 / ALXN2220.

33. 33. The pharmaceutical composition of any one of claims 1 to 32, wherein the antibody comprises two heavy chains and two light chains, each heavy chain consisting of 450 amino acid residues having SEQ ID NO: 9, and each light chain consisting of 214 amino acid residues having SEQ ID NO:

10.

34. 33. The pharmaceutical composition of any one of claims 1 to 32, wherein the antibody comprises two heavy chains and two light chains, each heavy chain consisting of 449 amino acid residues having SEQ ID NO: 13, and each light chain consisting of 214 amino acid residues having SEQ ID NO:

10.

35. 33. The pharmaceutical composition of any one of claims 1 to 32, wherein the antibody comprises two heavy chains and two light chains, each heavy chain consisting of 449 amino acid residues having SEQ ID NO: 14, and each light chain consisting of 214 amino acid residues having SEQ ID NO:

10.

36. 33. The pharmaceutical composition of any one of claims 1 to 32, wherein the antibody comprises two heavy chains and two light chains, each heavy chain consisting of 448 amino acid residues having SEQ ID NO: 15, and each light chain consisting of 214 amino acid residues having SEQ ID NO:

10.

37. The pharmaceutical composition according to any one of claims 1 to 36, wherein the antibody is produced in CHO host cells, preferably CHO-K1 cells, and purified from the cell culture.

38. 38. The pharmaceutical composition of any one of claims 27 to 33 and 37, wherein the heavy chain of the antibody comprises an N-terminal glutaminyl cyclization.

39. 39. The pharmaceutical composition of any one of claims 27 to 33 and 37 and 38, wherein the heavy chain comprises a C-terminal lysine clipping.

40. 40. An antibody for use in the method of any one of claims 27 to 39 which is N-glycosylated, preferably wherein the N-linked glycan is on N300 of the heavy chain.

41. 41. The pharmaceutical composition of any one of claims 1 to 40, wherein the anti-FTP antibody or antigen-binding fragment thereof is present at a concentration of about 1 mg / mL to about 500 mg / mL.

42. 43. The pharmaceutical composition of any one of claims 1 to 42, wherein the anti-FAP antibody or antigen-binding fragment thereof is present at a concentration of about 1 mg / mL to about 150 mg / mL.

43. 43. The pharmaceutical composition of any one of claims 1 to 42, wherein the anti-FTP antibody or antigen-binding fragment thereof is present at a concentration of about 50 mg / mL to about 100 mg / mL.

44. 44. The pharmaceutical composition of any one of claims 1 to 43, wherein the pharmaceutical composition is present in a volume of from about 0.1 mL to about 100 mL.

45. 45. The pharmaceutical composition of any one of claims 1 to 44, wherein the pharmaceutical composition is present in a volume of from about 1 mL to about 25 mL.

46. 46. ​​The pharmaceutical composition of any one of claims 1 to 45, wherein the pharmaceutical composition is present in a volume of from about 2 mL to about 20 mL.

47. The pharmaceutical composition of any one of claims 1 to 46, wherein the pharmaceutical composition is formulated for intravenous injection.

48. 48. The pharmaceutical composition of any one of claims 1 to 47, wherein the pharmaceutical composition has not been reconstituted from a lyophilized anti-FAP antibody or antigen-binding fragment thereof and / or has not been further lyophilized.

49. 49. The pharmaceutical composition of any one of claims 1 to 48, wherein the pharmaceutical composition is essentially free of sodium chloride.

50. 50. The pharmaceutical composition of any one of claims 1 to 49, wherein the pharmaceutical composition formulation is essentially free of poloxamer.

51. 51. The pharmaceutical composition of any one of claims 1 to 50, wherein the pharmaceutical composition meets at least one of the following stability criteria, and optionally any combination of two or all three of the stability criteria: remains stable at 40±2°C and 75±5% relative humidity (RH) for at least 1 week, preferably up to 1 month; remains stable at 25±2°C / 60±5% RH for at least 1 month, preferably up to 6 months; remains stable at 5±3°C for at least 1 month, preferably at least 18 months.

52. The pharmaceutical composition satisfies one, two, three or all four stability criteria (a)-(d): (a) the main peak is reduced by less than 1% by weight of the antibody under heat stress conditions at about 40°C for 4 weeks and / or at about 25°C for 12 weeks, as measured by size exclusion chromatography (SEC)-HPLC analysis; (b) the pharmaceutical composition exhibits a content of acidic species of the anti-FTP antibody or antigen-binding fragment thereof of less than 42.5% under heat stress conditions at about 40°C for 2 weeks, as measured by capillary isoelectric focusing (cIEF); (c) the pharmaceutical composition exhibits no substantial change in the content of acidic species of the anti-FTP antibody after three freeze-thaw cycles (from about −70° C. to room temperature), as measured by capillary isoelectric focusing (cIEF); and / or d) the anti-FAP antibody or antigen-binding fragment thereof retains at least 80% of its binding ability to a FAP protein after 4 weeks of storage at about 40°C and / or at least 70% of its binding ability to a FAP protein after 12 weeks of storage at about 25°C as measured by ELISA and compared to a control.

53. 53. The pharmaceutical composition of any one of claims 1 to 52, wherein the pharmaceutical composition exhibits a main peak ≧50.0%, an acidic peak ≦40.0%, and a basic peak ≦15.0%, e.g., as measured by cIEF; a main peak (monomer) ≧95.0% and high molecular weight species (HMWS) ≦5.0%, e.g., as measured by size exclusion chromatography (SEC)-HPLC analysis; a pH of 5.8±0.5; an osmolality of ≧240 mOsm / Kg; and an antibody concentration of 50±5.0 mg / mL.

54. The pharmaceutical composition satisfies one, two, three, or all four stability criteria (i)-(iv): (i) the main peak declines less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2% as measured by SEC-HPLC under heat stress conditions at about 40°C for 1 month, or at about 25°C for 6 months, or during long-term storage at about 5°C for 18 months; (ii) the pharmaceutical composition exhibits a content of acidic species of the anti-FTP antibody or antigen-binding fragment thereof of about 40% or less under heat stress conditions at about 40°C for 2 weeks or at about 25°C for 3 months, as measured by cIEF; (iii) the pharmaceutical composition exhibits a content of acidic species of the anti-FAP antibody or antigen-binding fragment thereof of less than 40%, preferably less than 35%, under long-term storage conditions of 12 or 18 months at about 5° C. as measured by cIEF; and / or (iv) the anti-FAP antibody or antigen-binding fragment thereof retains at least 80%, preferably at least 90%, of its ability to bind to a FAP protein after 6 months of storage at about 25°C, or after 12 or 18 months of storage at about 5°C, compared to a control, as measured by ELISA.

55. 55. The pharmaceutical composition of any one of claims 1 to 54, wherein the pharmaceutical composition has an osmolality of ≥ 240 mOsm / Kg.

56. 56. The pharmaceutical composition of any one of claims 1 to 55, wherein the pharmaceutical composition is sterile.

57. 57. The pharmaceutical composition of any one of claims 1 to 56, wherein the pharmaceutical composition is stable upon freezing and thawing.

58. 58. The pharmaceutical composition of any one of claims 1-57, wherein the pharmaceutical composition is present in one or more vials or infusion bottles containing a total of about 2500 mg to about 7500 mg of the anti-FAP antibody or antigen-binding fragment thereof.

59. 59. The pharmaceutical composition according to any one of claims 1 to 58, wherein the pharmaceutical composition is present in a vial or infusion bottle, preferably a vial or infusion bottle containing a volume of between 2 mL and 20 mL, such as a 2 mL or 20 mL type I clear glass vial.

60. 60. The pharmaceutical composition of claim 59, wherein the vial or infusion bottle contains an overfill of about 12.5% ​​volume, or a total volume of about 2.25 mL or 22.5 mL of the anti-FAP antibody or antigen-binding fragment thereof.

61. (a) the pharmaceutical composition comprises 6.5% (w / v) sucrose (65 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), pH 5.8, and a volume of 2 to 50 mL; and (b) the anti-FATTR antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO:7 and a VL region comprising the amino acid sequence of SEQ ID NO:

8.

62. (a) the pharmaceutical composition comprises 8% (w / v) sucrose (80 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), pH 5.8, and a volume of 2 to 50 mL; and (b) the anti-FATTR antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO:7 and a VL region comprising the amino acid sequence of SEQ ID NO:

8.

63. (a) the pharmaceutical composition comprises 6.5% (w / v) sucrose (65 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), pH 5.8, and a volume of 2 to 50 mL; and (b) the anti-FATTR antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11 and a VL region comprising the amino acid sequence of SEQ ID NO:

12.

64. (a) the pharmaceutical composition comprises 8% (w / v) sucrose (80 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), pH 5.8, and a volume of 2 to 50 mL; and (b) the anti-FATTR antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11 and a VL region comprising the amino acid sequence of SEQ ID NO:

12.

65. 1. A pharmaceutical composition comprising a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof at a concentration of about 50 mg / mL to about 150 mg / mL, a histidine buffer at a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80; 1. A pharmaceutical composition, wherein the anti-FAP antibody or antigen-binding fragment thereof is capable of binding to mutated, misfolded, misassembled, and / or aggregated FAP species and / or fragments thereof, and does not substantially recognize physiological FAP species.

66. 1. A pharmaceutical composition formulated for intravenous injection comprising 50 mg / mL of a human anti-transthyretin (TTR) antibody comprising a heavy chain variable (VH) region having the amino acid sequence of SEQ ID NO: 7 or 11 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 8 or 12, wherein the pharmaceutical composition comprises 6.5% weight per unit volume (w / v) sucrose (65 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), and has a pH of 5.

8.

67. 67. The pharmaceutical composition of claim 66, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

68. 67. The pharmaceutical composition of claim 66, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

69. 67. The pharmaceutical composition of claim 66, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

70. 70. The pharmaceutical composition of any one of claims 66 to 69, wherein the pharmaceutical composition has a volume of 2 ml.

71. 70. The pharmaceutical composition of any one of claims 66 to 69, wherein the pharmaceutical composition has a volume of 20 ml.

72. 1. A pharmaceutical composition formulated for intravenous injection comprising 50 mg / mL of a human anti-FTP antibody comprising a VH region having the amino acid sequence of SEQ ID NO:7 or 11 and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 12, the pharmaceutical composition comprising 8% (w / v) sucrose (8 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), and having a pH of 5.

8.

73. 73. The pharmaceutical composition of claim 72, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

74. 73. The pharmaceutical composition of claim 72, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

75. 73. The pharmaceutical composition of claim 72, wherein the VH comprises the amino acid sequence of SEQ ID NO: 8 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

76. 76. The pharmaceutical composition of any one of claims 72 to 75, wherein the pharmaceutical composition has a volume of 2 ml.

77. 66. The pharmaceutical composition of any one of claims 72 to 65, wherein the pharmaceutical composition has a volume of 20 ml.

78. 1. A pharmaceutical composition formulated for intravenous injection comprising approximately 50 mg / ml of a human anti-transthyretin (TTR) antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:9 and each light chain having the amino acid sequence of SEQ ID NO:10; 6.5% weight per unit volume (w / v) sucrose (65 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), and having a pH of 5.

8.

79. 79. The pharmaceutical composition of claim 78, wherein the heavy chain of the antibody lacks a C-terminal lysine, the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

80. 80. The pharmaceutical composition of claim 79, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

81. 81. The pharmaceutical composition of any one of claims 78 to 80, wherein the pharmaceutical composition has a volume of 2 ml.

82. 81. The pharmaceutical composition of any one of claims 78 to 80, wherein the pharmaceutical composition has a volume of 20 ml.

83. 1. A pharmaceutical composition formulated for intravenous injection comprising approximately 50 mg / ml of a human anti-transthyretin (TTR) antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO: 9 and each light chain having the amino acid sequence of SEQ ID NO: 10; the pharmaceutical composition comprising 8% weight per unit volume (w / v) sucrose (80 mg / mL sucrose), 0.03% (w / v) polysorbate 80 (0.3 mg / mL polysorbate 80), 20 mM histidine (1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monohydrochloride), and having a pH of 5.

8.

84. 84. The pharmaceutical composition of claim 83, wherein the antibody heavy chain lacks a C-terminal lysine, the N-terminal glutamine of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

85. 85. The pharmaceutical composition of claim 84, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

86. 86. The pharmaceutical composition of any one of claims 83 to 85, wherein the pharmaceutical composition has a volume of 2 ml.

87. 86. The pharmaceutical composition of any one of claims 83 to 85, wherein the pharmaceutical composition has a volume of 20 ml.

88. A kit comprising the pharmaceutical composition of any one of claims 1 to 87 and instructions for its use.

89. A pharmaceutical composition according to any one of claims 1 to 87 or a kit according to claim 88 for use in the treatment or prevention of transthyretin-mediated amyloidosis (ATTR) in a human subject.

90. 88. A method of treating ATTR in a human subject, comprising administering to the human subject a pharmaceutical composition according to any one of claims 1 to 87.

91. 88. A method of treating ATTR associated cardiomyopathy (ATTR-CM) in a human subject, comprising administering to the human subject the pharmaceutical composition of any one of claims 1 to 87.

92. 1. A method of treating transthyretin-mediated amyloidosis (ATTR) in a human subject, comprising administering 50 mg / ml of a pharmaceutical composition comprising a human anti-FTR antibody comprising a VH region having the amino acid sequence of SEQ ID NO:7 or 11 and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 12, wherein the pharmaceutical composition comprises 6.5% (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine, and has a pH of 5.

8.

93. 93. The method of claim 92, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

94. 93. The method of claim 92, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

95. 93. The method of claim 92, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

96. 96. The method of any one of claims 92-95, wherein the method comprises administering to the subject the pharmaceutical composition at a dose that provides 30-60 mg / kg of the human anti-FAP antibody or up to 7500 mg of the human anti-FAP antibody.

97. 1. A method of treating ATTR in a human subject comprising administering 50 mg / ml of a pharmaceutical composition comprising a human anti-FTR antibody comprising a VH region having the amino acid sequence of SEQ ID NO:7 or 11 and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 12, wherein the pharmaceutical composition comprises 8% (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine, and a pH of 5.

8.

98. 98. The method of claim 97, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

99. 98. The method of claim 97, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

100. 98. The method of claim 97, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

101. 101. The method of any one of claims 97-100, wherein the method comprises administering to the subject the pharmaceutical composition at a dose that provides 30-60 mg / kg of the human anti-FAP antibody or up to 7500 mg of the human anti-FAP antibody.

102. 1. A method of treating ATTR in a human subject comprising administering about 50 mg / ml of a pharmaceutical composition comprising a human anti-FAT antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:9 and each light chain having the amino acid sequence of SEQ ID NO:10, wherein the pharmaceutical composition comprises 6.5% weight per unit volume (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine, and has a pH of 5.

8.

103. 103. The method of claim 102, wherein the heavy chain of the antibody lacks a C-terminal lysine, the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

104. 104. The method of claim 103, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

105. 105. The method of any one of claims 102 to 104, wherein the method comprises administering to the subject the pharmaceutical composition at a dose that provides 30-60 mg / kg of the human anti-FAP antibody or up to 7500 mg of the human anti-FAP antibody.

106. 1. A method of treating ATTR in a human subject comprising administering about 50 mg / ml of a pharmaceutical composition comprising a human anti-FAT antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:9 and each light chain having the amino acid sequence of SEQ ID NO:10, wherein the pharmaceutical composition comprises 8% weight per unit volume (w / v) sucrose, 0.03% (w / v) polysorbate 80, 20 mM histidine, and a pH of 5.

8.

107. 107. The method of claim 106, wherein the heavy chain of the antibody lacks a C-terminal lysine, the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

108. 108. The method of claim 107, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

109. 109. The method of any one of claims 106-108, wherein the method comprises administering to the subject the pharmaceutical composition at a dose that provides 30-60 mg / kg of the human anti-FAP antibody or up to 7500 mg of the human anti-FAP antibody.

110. 88. A pharmaceutical product comprising the pharmaceutical composition of any one of claims 1 to 87 for use in a method for treating ATTR in a subject in need thereof, preferably for use in treating a subject with ATTR amyloidosis with cardiomyopathy (ATTR-CM).

111. 111. A sterile pharmaceutical container comprising the pharmaceutical composition of any one of claims 1 to 87 or the pharmaceutical product of claim 110, preferably said container being a disposable glass vial, preferably sealed with a rubber stopper and an aluminium-plastic cover flip-off cap, or an infusion bottle, or an infusion bag, or a dosing syringe in a dosing pump, containing about 100 mg of antibody at a concentration of about 50±5 mg / mL to about 150 mg / mL, such as about 100 mg / mL, optionally wherein said vial comprises an overfill of approximately 10% by volume.

112. 112. The pharmaceutical product of claim 110 or the container of claim 111, wherein the pharmaceutical composition is ready for administration to a subject in need thereof, preferably by intravenous infusion.

113. 113. A pharmaceutical product or container as described in claim 112, wherein the pharmaceutical composition is diluted before injection in a solution of glucose or a polymer thereof, preferably wherein the polymer is dextran.

114. 114. The pharmaceutical product or container of claim 113, wherein the pharmaceutical composition is diluted to a concentration of about 1 mg / mL to about 50 mg / mL.

115. 115. A pharmaceutical product or container as described in claim 113 or 114, wherein the concentration of glucose or a polymer thereof is 5% (w / v).

116. 1. Use of a pharmaceutical composition in the manufacture of a medicament for treating or preventing ATTR in a subject, the pharmaceutical composition comprising: (a) a human anti-FAT receptor antibody or antigen-binding fragment thereof capable of binding to aggregated FAT receptor species and not substantially recognizing physiological FAT receptor species; (b) one or more of sucrose, polysorbate 80, and a polar excipient.

117. 117. The use of claim 116, wherein the human anti-FTP antibody or antigen-binding fragment thereof is present in the medicament at a concentration of about 50 mg / mL to about 150 mg / mL, and the medicament comprises a histidine buffer at a pH of about 5.8, 6.5% or 8.0% (w / v) sucrose, and 0.03% (w / v) polysorbate 80.

118. The use according to claim 116 or 117, wherein the pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 87.

119. 1. Use of a pharmaceutical composition in the manufacture of a medicament for treating ATTR in a human subject, the pharmaceutical composition comprising approximately 50 mg / ml of a human anti-FATTR antibody comprising a VH region having the amino acid sequence of SEQ ID NO:7 or 11 and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 12, the pharmaceutical composition comprising 6.5% (w / v) sucrose; 0.03% (w / v) polysorbate 80; and 20 mM histidine, and having a pH of 5.

8.

120. The use described in claim 119, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

121. The use described in claim 119, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

122. The use of claim 100, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

123. 1. Use of a pharmaceutical composition in the manufacture of a medicament for treating ATTR in a human subject, the pharmaceutical composition comprising approximately 50 mg / ml of a human anti-FATTR antibody comprising a VH region having the amino acid sequence of SEQ ID NO:7 or 11 and a VL region comprising the amino acid sequence of SEQ ID NO:8 or 12, the pharmaceutical composition comprising 8% (w / v) sucrose; 0.03% (w / v) polysorbate 80; and 20 mM histidine, and having a pH of 5.

8.

124. The use of claim 123, wherein the VH comprises the amino acid sequence of SEQ ID NO: 7 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

125. The use of claim 123, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

8.

126. The use of claim 123, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO:

12.

127. 1. Use of a pharmaceutical composition in the manufacture of a medicament for treating ATTR in a human subject, the pharmaceutical composition comprising 50 mg / ml of a human anti-FATTR antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:9 and each light chain having the amino acid sequence of SEQ ID NO:10, the pharmaceutical composition comprising 6.5% (w / v) sucrose; 0.03% (w / v) polysorbate 80; and 20 mM histidine, at a pH of 5.

8.

128. The use of claim 127, wherein the heavy chain of the antibody lacks a C-terminal lysine, the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

129. 129. The use of claim 128, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

130. 1. Use of a pharmaceutical composition in the manufacture of a medicament for treating ATTR in a human subject, the pharmaceutical composition comprising 50 mg / ml of a human anti-FATTR antibody comprising two heavy chains and two light chains, each heavy chain having the amino acid sequence of SEQ ID NO:9 and each light chain having the amino acid sequence of SEQ ID NO:10, the pharmaceutical composition comprising 8% (w / v) sucrose; 0.03% (w / v) polysorbate 80; and 20 mM histidine, at a pH of 5.

8.

131. The use of claim 108, wherein the heavy chain of the antibody lacks a C-terminal lysine, the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, and the heavy chain is N-glycosylated.

132. 132. The use of claim 131, wherein the heavy chain has the amino acid sequence of SEQ ID NO:

15.

133. 116. An article of manufacture comprising one or more containers according to any one of claims 111 to 115 and a label or package insert, the label or package insert specifying that the antibody is indicated for the treatment of ATTR, particularly wild-type or hereditary transthyretin-mediated amyloid cardiomyopathy (ATTR-CM).

134. 134. The article of manufacture of claim 133, wherein the formulation of the antibody is provided as a concentrate for infusion solution, preferably presented as a clear to milky white and colorless to pale yellow solution without preservatives, provided as 100 mg / 2 mL in a vial.

135. 1. A method for producing a pharmaceutical composition of a human anti-transthyretin (TTR) antibody or antigen-binding fragment thereof capable of binding to aggregated TTR species and not substantially recognizing physiological TTR species, the method comprising formulating the antibody in a buffer comprising sucrose and / or polysorbate 80.

136. The method of claim 135, wherein the pharmaceutical composition is a pharmaceutical composition according to any one of claims 1 to 87.

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