Pharmaceutical composition for treating or preventing transthyretin-mediated amyloidosis

Abstract

Transthyretin (TTR) is a soluble protein involved in the transport of thyroxine and retinol in vivo. Under certain conditions, the TTR proteins employ misfolded, misassembled, and / or aggregated TTR conformations and become toxic, which may lead to transthyretin-mediated amyloidosis (ATTR). Provided herein, inter alia, are compositions (e.g., pharmaceutical compositions) and related articles of manufacture comprising anti-TTR antibodies or antigen fragments thereof. In addition, provided herein are, inter alia, methods of treating or preventing ATTR using the pharmaceutical compositions described herein.

Description

[0001] This application claims the priority benefits of European Application No. EP 22 207 645.7, US Provisional Application No. 63 / 383,803, both filed on November 15, 2022, and US Provisional Application No. 63 / 503,286 filed on May 19, 2023.

[0002] The entire contents of the above-mentioned patent applications are incorporated herein by reference. Technical Field

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

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

[0005] Under certain conditions that have not been fully elucidated and may include acidic pH, oxidative stress, and local factors, the TTR protein adopts a misfolded, misassembled, and / or aggregated TTR conformation and becomes toxic, which can lead to transthyretin-mediated amyloidosis (ATTR).

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

[0007] The present disclosure particularly provides compositions (e.g., pharmaceutical compositions) and related articles containing an anti-transthyretin (TTR) antibody or an antigenic fragment thereof as a medicament. Additionally, the present disclosure particularly provides methods of using the pharmaceutical compositions described herein to treat or prevent transthyretin-mediated amyloidosis (ATTR) in a subject in need thereof.

[0008] According to the present invention, a pharmaceutical preparation of a recombinant human monoclonal antibody specific for the disease-related amyloidogenic form of TTR, having ALXN2220, also known as NI006, at a concentration of about 25 mg / ml to about 150 mg / ml, typically about 50 mg / ml, is developed. The pharmaceutical preparation is characterized in that the pH is 5.3 to 6.3, preferably pH 5.8 ± 0.1, and generally contains a histidine buffer and sucrose and polysorbate, preferably polysorbate 80, as excipients, as well as water for infusion / injection. The preparation of the present invention allows the antibody to be stored in liquid for a long time without lyophilization, and is intravenously administered to a subject in need thereof. In fact, the antibody preparation of the present invention has been successfully used in the first-in-human study of NI006 in patients with transthyretin amyloid cardiomyopathy (ATTR-CM), in which the preparation has been administered at 0.3 to 60 mg / kg body weight without any drug-related serious adverse events and without any adverse events caused by the preparation; 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 preparations of two murine monoclonal anti-TTR antibodies named 9D5 and 14G8 and their humanized forms, wherein the most preferred preparation (Preparation F25) contains about 50 mg / ml of humanized antibody 14G8, 20 mM histidine, 240 mM sucrose and 0.04% poloxamer 188 (PX188), and the pH is 6.0; see paragraph

[00269] on page 57 of WO 2019 / 108689 A1. It is worth noting that WO 2019 / 108689 A1 also describes the study of other buffers and excipients, including polysorbate 20 (PS20) and polysorbate (PS80), and specifically excludes preparations containing, for example, a combination of sucrose and PS80. In addition, WO 2019 / 108689 A1 only provides two short-term experiments (one week at 50 °C and two weeks at 25 °C) of the test preparation in liquid, while the experiments of "long-term storage" (one month and three months) at different temperatures are carried out with the lyophilized preparation.

[0010] In contrast, the present invention generally relates to liquid formulations of anti-TTR antibodies having a long shelf life, which have the advantages of being ready-to-use without reconstitution, reducing the risk of errors due to the absence of a reconstitution step, and faster administration, which is particularly beneficial in acute settings.

[0011] In a preferred embodiment, the solution to this objective as characterized in the claims is illustrated in the appended examples, wherein the formulation consists essentially of the anti-TTR antibody ALXN2220 (also referred to herein as NI006), a histidine buffer, and sucrose and polysorbate 80 (PS80) as excipients, with a pH of about 5.8 and an antibody concentration of about 50 mg / ml or about 100 mg / ml.

[0012] Specifically, in the examples, a 50 mg / ml antibody formulation (pH 5.8) in 20 mM histidine, 6.5% or 8% (w / v) sucrose, and 0.03% (w / v) PS80 was identified as a lead candidate for further development and clinical use and thus represents the most preferred embodiment of the present invention and an equivalent formulation that maintains the physicochemical properties crucial for the stability and function of the antibody as tested and verified in the appended examples. To vary the components in the preferred formulation and / or the concentration of the excipients, the following factors should be considered to ensure the stability and efficacy of the antibody:

[0013] Histidine: Used as a buffer. The buffering capacity is related to the ability of histidine to maintain the pH close to its pKa value. Since the pKa of histidine is approximately 6.0 at pH 5.8, its efficiency is slightly lower, but it can still be used as a buffer. The buffering capacity is also affected by the concentration of the buffer and the proximity of the pH to the pKa.

[0014] Sucrose: Stabilizes proteins during freeze / thaw cycles and increases the osmolality. Sucrose significantly contributes to the osmolality of the solution, which is important for maintaining the structure of IgG antibodies.

[0015] High concentration of the target protein: Like all proteins, antibodies have a buffering capacity due to the presence of ionizable groups in their amino acid side chains. These groups include the carboxyl groups of aspartic and glutamic acids, the amino groups of lysine and arginine, the imidazole group of histidine, the hydroxyl group of tyrosine, the thiol group of cysteine, and the terminal amino and carboxyl groups.

[0016] pH 5.8: The pH is slightly lower than the pKa of histidine, which will still provide a buffering effect.

[0017] For example, if the initial 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 the protonated form (HA) and the deprotonated form (A-) can be doubled to 40 mM. Since changing the total concentration of the buffer will affect the ionic strength and osmolality of the solution, which may have an impact on the stability and solubility of the protein, the concentration and / or nature of the osmolyte (sucrose in this case) can be adjusted and the corresponding formulations can be tested according to the examples.

[0018] The parental antibody of NI006 was first described in WO 2015 / 092077 A1 (named antibody NI-301.37F1) and Michalon et al., Nat. Commun. 12 (2021), 3142 (named antibody NI301A). As disclosed in WO 2015 / 092077 A1, NI006 (NI-301.37F1) is particularly characterized by binding to aggregated human wild-type transthyretin (wtATTR), which is shown in Figures 2 to 4 and Figure 7, and described in Examples 3 to 6, and further described in the last paragraph on page 46. In addition, WO 2015 / 092077 A1 discloses that NI006 (NI-301.37F1) does not bind to the monomer and dimer of human native transthyretin (TTR), as shown in Example 5 and Figure 4 shown. This binding characteristic is advantageous because the antibody selectively binds to aggregated wtTTR, and thus initially not only considering the treatment of hereditary transthyretin amyloidosis (hATTR) with polyneuropathy (previously called familial amyloid polyneuropathy, FAP) due to mutations in the gene encoding TTR, but also considering the treatment of wild-type transthyretin amyloidosis (wtATTR) (called senile systemic amyloidosis (SSA)). In addition, there is no risk that the antibody interferes with the assembly of native monomers into physiological tetramers. Therefore, in a preferred embodiment of the pharmaceutical antibody formulation of the present invention, the antibody is ALXN2220 / NI006 or an equivalent antibody that substantially has the TTR binding characteristics of NI006 and is preferably human. For example, WO 2015 / 092077 A1 discloses two other human antibodies (i.e., antibodies NI-301.59F1 and NI-301.35G11) that exhibit the said binding characteristics, as well as two human antibodies NI-301.28B3 and NI301.12D3 that have substantially the same epitope as NI006 (NI-301.37F7).

[0019] Although human antibodies are particularly preferred, for example because they are inherently less likely to elicit an anti-drug antibody (ADA) response, the formulations of the present invention include humanized antibodies and human sequence monoclonal antibodies from animals such as mice. Additionally, as previously described, the subject formulations have been shown to be safe and effective; see ClinicalTrials.gov ID NCT04360434 and Garcia-Pavia et al., (2023), supra.

[0020] As exemplified in the appended examples, several stability tests performed with the formulations and pharmaceutical compositions of the present invention have demonstrated that the antibody remains stable for at least 1 month under various conditions, such as at 40 ± 2 °C and 75 ± 5% relative humidity (RH) (stress stability study); remains stable for at least 6 months at 25 ± 2 °C / 60 ± 5% RH (accelerated stability study); and remains stable for at least 12 months to 18 months at 5 ± 3 °C (long-term stability study).

[0021] Accordingly, the present invention relates to a pharmaceutical composition (also referred to herein as a pharmaceutical formulation) that comprises a human anti-transthyretin (TTR) antibody or an antigen-binding fragment thereof that is capable of binding to mutant, misfolded, misassembled, and / or aggregated TTR material, specifically aggregated human TTR material and / or fragments thereof, and that substantially does not recognize physiological TTR material, wherein the pharmaceutical composition comprises one or more of sucrose, a polysorbate (preferably polysorbate 80), and a polar excipient (e.g., histidine, such as L-histidine and / or L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof), preferably wherein the antibody remains stable for at least 1 week, preferably up to 1 month, at 40 ± 2 °C and 75 ± 5% RH; remains stable for at least 1 month, and preferably up to 6 months, at 25 ± 2 °C / 60 ± 5% RH; and / or remains stable for at least 1 month, preferably up to 12 months, more preferably up to 18 months, at 5 ± 3 °C. In a preferred embodiment, the pharmaceutical composition is an aqueous composition, also referred to as a liquid formulation, and thus also comprises water for injection.

[0022] The polar excipient included in the pharmaceutical composition of the present invention is also referred to as a buffer system, preferably a histidine buffer (e.g., consisting of L-histidine and L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof).

[0023] In a preferred embodiment, the polar excipient (e.g., histidine such as L-histidine and / or L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof) is present in the pharmaceutical composition of the present invention in an amount of from about 1 mM to about 100 mM (e.g., from 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., from 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., from 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., L-histidine and / or L-histidine monohydrochloride or a pharmaceutically acceptable salt thereof, preferably provided by 1.06 mg / mL L-histidine and 2.78 mg / mL L-histidine monochloride).

[0024] In addition or alternatively, the pharmaceutical composition of the present invention comprises about 6% to about 9% (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5% or 9%) weight / unit volume (w / v) of sucrose.

[0025] Preferably, the pharmaceutical composition of the present invention comprises about 6% to about 7% (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%) w / v sucrose, and more preferably, the pharmaceutical composition of the present invention comprises about 6.5% w / v sucrose.

[0026] Alternatively, the pharmaceutical composition of the present invention comprises 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 composition of the present invention comprises about 8% w / v sucrose.

[0027] In addition, in any of the foregoing embodiments, the pharmaceutical composition of the present invention comprises from about 0.001% to about 0.1% w / v (such as from about 0.001% to about 0.01%, such as about 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009% or 0.01%, such as from about 0.01% to about 0.1%, such as about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%) of polysorbate, preferably polysorbate 80.

[0028] More preferably, the pharmaceutical composition of the present invention comprises from about 0.01% to about 0.05% w / v (such as, 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 comprises about 0.03% w / v of polysorbate 80.

[0029] In a preferred embodiment, the pharmaceutical composition of the present invention has a pH of from about 5.3 to about 6.3 (such as from about 5.3 to about 5.8, such as about 5.3, 5.4, 5.5, 5.6, 5.7 or 5.8, such as from about 5.5 to about 6.0, such as about 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0, such as from about 5.8 to about 6.3, such as about 5.8, 5.9, 6.0, 6.1, 6.2 or 6.3).

[0030] Most preferably, the pharmaceutical composition of the present invention has a pH of about 5.8.

[0031] In principle, the antibody comprised in the pharmaceutical composition can be any anti-TTR antibody that recognizes the amyloidogenic form of TTR (i.e., the aggregated TTR species), and preferably human aggregated TTR, but does not bind to the physiological TTR species. In a particularly preferred embodiment, the anti-TTR antibody for use in the pharmaceutical composition of the present invention is NI006 / ALXN2220 or an equivalent antibody derived from the human antibody NI-301.37F1, as characterized in WO 2015 / 092077 A1 and Michalon et al., Nat Commun. 12 (2021), 3142; see also ibid.

[0032] Thus, in a preferred embodiment, the anti-TTR antibody or antigen-binding fragment thereof comprised in the pharmaceutical composition of the present invention comprises a heavy-chain variable region (VH) having the three complementarity-determining regions (CDRs) shown in SEQ ID NOs: 1-3 and a light-chain variable region (VL) having the three CDRs shown in SEQ ID NOs: 4-6.

[0033] 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) with SEQ ID NO:7, and the VL comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity) with SEQ ID NO:8.

[0034] 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) with SEQ ID NO:11, and the VL comprises an amino acid sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity) with SEQ ID NO:12.

[0035] Preferably, the VH region comprises the amino acid sequence of SEQ ID NO:7 and the VL comprises the amino acid of SEQ ID NO:8, or the VH region comprises the amino acid sequence of SEQ ID NO:11 and the VL comprises the amino acid of SEQ ID NO:12.

[0036] In some embodiments, the VH region of the anti-TTR antibody or its antigen-binding fragment comprises 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 sequences 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 its antigen-binding fragment 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 sequences of SEQ ID NO:4, SEQ ID NO:5 and / or SEQ ID NO:6. In some aspects, the VH region of the anti-TTR antibody or its antigen-binding fragment comprises one or more CDR sequences comprising an amino acid sequence having 1, 2 or 3 mismatches relative to the amino acid sequences 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 its antigen-binding fragment comprises one or more CDR sequences comprising an amino acid sequence having 1, 2 or 3 mismatches relative to the amino acid sequences of SEQ ID NO:4, SEQ ID NO:5 and / or SEQ ID NO:6.

[0037] To avoid the production of "anti-drug antibodies" (ADA) in subjects administered the antibodies or antigen-binding fragments described herein, the antibodies are preferably human or humanized antibodies, typically human IgG, and most preferably human IgG1. In a preferred embodiment, the antibody is a human IgG1m3 allotype. Thus, preferably, 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, as characterized in WO 2015 / 092077A1 and Michalon et al., Nat Commun. 12 (2021), 3142. The antibody NI006 / ALXN2220 is a fully human IgG1m3 allotype antibody and thus comprises the human constant heavy chain (HC) amino acid sequence present in SEQ ID NO:9 and the corresponding human constant light chain (LC), with the κ light chain exemplified as in SEQ ID NO:10. As further described below, the IgG antibody is configured as a tetramer consisting of an HC and two LC light chains linked by disulfide bridges.

[0038] The antibody NI006 / ALXN2220 can be produced in Chinese hamster ovary (CHO) cells. CHO cells are the most widely used mammalian cells for producing recombinant monoclonal antibodies due to their ability to perform post-translational modifications (PTMs) on antibody molecules, which also typically occur in the human body. Through genetic manipulation by mutagenesis, different CHO sub-cells with improved quality have been established. These variants include CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Thus, in one embodiment, the antibody for 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.

[0039] As shown in Example 3, the major PTMs identified in the antibody NI006 / ALXN2220 are the modification of the N-terminal glutamine to pyroglutamic acid in the HC, the deletion of the C-terminal lysine, and N-glycosylation. In this context, the N-glycosylation site was identified at position Asn300 (HC N300, SEQ ID NO:9).

[0040] Thus, in one embodiment, the heavy chain of the anti-TTR antibody present in the pharmaceutical composition of the present invention has a deleted C-terminal lysine, i.e., the antibody has undergone C-terminal lysine cleavage. Specifically, the C-terminal lysine as shown in SEQ ID NO:9 is cleaved from the heavy chain of the antibody, preferably from each heavy chain of the antibody. The sequence (i.e., the heavy chain sequence with the cleaved C-terminal lysine) is shown in SEQ ID NO:13. Alternatively, the N-terminal glutamine is modified to pyroglutamic acid, i.e., the heavy chain of the antibody as shown in SEQ ID NO:9 has undergone N-terminal glutamine acylation cyclization. The sequence (i.e., the heavy chain sequence containing cyclic pyroglutamic acid and lacking the N-terminal glutamine) is shown in SEQ ID NO:14.

[0041] Alternatively, the heavy chain of the anti-TTR antibody present in the pharmaceutical composition of the present invention has a deleted C-terminal lysine and the N-terminal glutamine is modified to pyroglutamic acid. The sequence (i.e., the heavy chain sequence with the cleaved C-terminal lysine and containing cyclic pyroglutamic acid and lacking the N-terminal glutamine) is shown in SEQ ID NO:15.

[0042] In addition or alternatively, the antibody is glycosylated, specifically N-glycosylated. More specifically, the heavy chain of the antibody is glycosylated, and even more specifically glycosylated at N300 of the heavy chain.

[0043] In a preferred embodiment, the anti-TTR antibody present in the pharmaceutical composition of the present invention lacks a C-terminal lysine, has a glutamine at the N-terminus modified to 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 above, wherein the antibody consists of two heavy chains having SEQ ID NO:9 and two light chains having SEQ ID NO:10, and wherein in the heavy chain, the N-terminal glutamine is modified to pyroglutamic acid, the C-terminal lysine is absent, 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 above, wherein the antibody consists of two heavy chains having SEQ ID NO:15 and two light chains having SEQ ID NO:10, and wherein the heavy chain is N-glycosylated.

[0044] 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 the heavy chain, and about 96% of the antibodies have a deletion of the C-terminal lysine. Thus, in one embodiment, about 99% of the antibodies in the formulation of the present invention have a heavy chain in which the N-terminal pyroglutamic acid is derived from N-terminal glutamine, and / or about 96% of the antibodies have a deletion of the C-terminal lysine.

[0045] In some embodiments, the anti-TTR antibody or its antigen-binding fragment is present in the pharmaceutical composition of the present invention at a concentration of about 1 mg / mL to about 500 mg / mL (such as about 1 mg / mL to about 100 mg / mL, such as 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, such as about 100 mg / mL to about 200 mg / mL, such as 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, such as about 200 mg / mL to about 300 mg / mL, such as 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, such as about 300 mg / mL to about 400 mg / mL, such as 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, such as about 400 mg / mL to about 500 mg / mL, such as 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).

[0046] In a preferred embodiment, 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 (such as about 1 mg / mL to about 20 mg / mL, such as about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL or about 20 mg / mL, such as about 20 mg / mL to about 40 mg / mL, such as 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, such as 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, such as about 60 mg / mL to about 80 mg / mL, such as about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL or about 80 mg / mL, such as about 80 mg / mL to about 100 mg / mL, such as 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 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 a volume of about 2 mL or 20 mL).

[0047] 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.

[0048] As mentioned above, the anti-TTR antibody or antigen-binding fragment thereof comprised 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 of the amino acid sequence of SEQ ID NO:7 or 11 and a light chain variable region of the amino acid of SEQ ID NO:8. In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof does not elicit an anti-drug antibody (ADA) response.

[0049] In some embodiments, the pharmaceutical composition of the present invention is present in a volume of from about 0.1 mL to about 100 mL (such as from about 0.1 mL to about 20 mL, such as 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, such as from about 20 mL to about 40 mL, such as about 20 mL, about 22.5 mL, about 25 mL, about 30 mL, about 35 mL or about 40 mL, such as from about 40 mL to about 60 mL, such as about 40 mL, about 45 mL, about 50 mL, about 55 mL or about 60 mL, such as from about 60 mL to about 80 mL, such as about 60 mL, about 65 mL, about 70 mL, about 75 mL or about 80 mL, such as from about 80 mL to about 100 mL, such as about 80 mL, about 85 mL, about 90 mL, about 95 mL or about 100 mL).

[0050] Preferably, the pharmaceutical composition of the present invention is present in a volume of from about 1 mL to about 10 mL (such as from about 1 mL to about 2 mL, such as 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, such as from about 1.5 mL to about 2.5 mL, such as 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, such as from about 2 mL to about 2.25 mL, such as about 2.1 mL, about 2.15 mL, about 2.2 mL or about 2.25 mL, such as from about 2 mL to about 5 mL, such as 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, such as from about 5 mL to about 10 mL, such as 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).

[0051] 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).

[0052] 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).

[0053] Most preferably, the pharmaceutical composition of the present invention is present in a volume of about 2 mL or about 20 mL.

[0054] In addition, the compatibility of the antibody formulation with materials used in clinical practice was evaluated. Specifically, when glucose or saline was used as a diluent, the compatibility with polyvinyl chloride IV bags, IV tubing, and filters, as well as with polyvinyl chloride syringes and corresponding non-polyvinyl chloride materials, 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 of Example 2 indicate that the formulation was generally compatible with the materials used in clinical practice evaluated. Specifically, in the saline group, visible particles were observed, indicating that NI006 / ALXN2220 was less stable in saline, where the data showed that no significant changes in appearance, protein concentration, subvisible particles, SEC-HPLC, and ELISA binding assays were observed when diluted in glucose solution. Thus, NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL was shown to be stable for 24 hours at 2 - 8 °C and then for 6 hours at 25 °C (for a total of 30 hours) and was compatible with the materials used in clinical practice evaluated.

[0055] The most common route of administration of monoclonal antibodies in therapy is intravenous (IV) infusion. This method is preferred because it allows the antibody to be delivered directly into the bloodstream, ensuring immediate distribution throughout the body and enabling precise dosing.

[0056] For monoclonal antibodies, intravenous administration is particularly important because of their large molecular size, which generally prevents their efficient absorption through the intestine or skin. This means that oral or transdermal delivery methods are not suitable for these types of drugs. Additionally, IV administration bypasses the first-pass metabolism in the liver, which can significantly alter the efficacy and safety of the drug.

[0057] Accordingly, the formulations of the present invention are developed especially for IV administration. Thus, in a preferred embodiment, the formulations of the present invention are suitable for intravenous administration.

[0058] 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.

[0059] Since in the formulations of the present invention, sucrose is used as a tonicity modifier and additional stabilizer for the antibody, there is no need for NaCl, especially when the main purpose of NaCl is to stabilize proteins. Sucrose can achieve this purpose without increasing the ionic strength of the solution. Therefore, the pharmaceutical compositions of the present invention are preferably substantially free of sodium chloride.

[0060] In addition or alternatively, the pharmaceutical compositions of the present invention are substantially free of (or, for example, completely lacking in) poloxamer.

[0061] In some embodiments, the pharmaceutical compositions of the present invention are characterized by any combination of one, two, three, or all four stability criteria (a) to (d): (a) the main peak decrease under heat stress conditions at about 40 °C for 4 weeks and / or at about 25 °C for 12 weeks is less than 1% by weight of the antibody, as measured by size exclusion chromatography (SEC)-HPLC analysis; (b) the pharmaceutical composition shows that the content of acidic species of the anti-TTR antibody under heat stress conditions at about 40 °C for 2 weeks is less than 42.5%, as measured by capillary isoelectric focusing (cIEF); (c) after 3 cycles of freeze-thaw (about -70 °C to room temperature), the pharmaceutical composition shows no significant change in the content of acidic species of the anti-TTR antibody, as measured by capillary isoelectric focusing (cIEF); and / or (d) the anti-TTR antibody retains at least 80% of its binding efficacy to TTR protein after storage at about 40 °C for 4 weeks and / or at least 70% of its binding efficacy to TTR protein after storage at about 25 °C for 12 weeks, for example, as measured by ELISA and relative to a control (e.g., not stored for a long time).

[0062] In some embodiments, the pharmaceutical composition shows a main peak ≥50.0%, an acidic peak ≤40.0%, and a basic peak ≤15.0% as measured by, for example, cIEF, a main peak (monomer) ≥95.0% and high molecular weight species (HMWS) ≤5.0% as measured by, for example, size exclusion chromatography (SEC)-HPLC analysis, a pH of 5.8 ± 0.5, an osmolality ≥240 mOsm / Kg, and an antibody concentration of 50 ± 5.0 mg / mL.

[0063] In addition, the pharmaceutical composition of the present invention may be characterized by any combination of one, two, three, or all four stability criteria (i) to (iv): (i) the main peak decline (representing monomer content) during 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 is less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2%, as measured by SEC-HPLC; (ii) the pharmaceutical composition shows that the content of acidic substances of the anti-TTR antibody is less than or approximately equal to about 40% 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 shows that the content of acidic substances of the anti-TTR antibody is less than 40%, preferably less than 35%, under long-term storage conditions at about 5 °C for 12 months 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 efficacy to the TTR protein after storage at about 25 °C for 6 months or after storage at about 5 °C for 12 months or 18 months, as measured by ELISA and relative to a control (e.g., not stored for a long time). Accordingly, the pharmaceutical composition may be referred to as a stable formulation. As illustrated in Example 2, the shelf life of the pharmaceutical product is currently set at 24 months when stored in the dark at 5 ± 3 °C. Accordingly, in one embodiment, the pharmaceutical composition (i.e., the formulation) has a shelf life of 24 months at 2 °C - 8 °C in the dark.

[0064] In addition or alternatively, the pharmaceutical composition of the present invention has an osmolality of ≥240 mOsm / Kg and comprises sucrose and an optional surfactant.

[0065] In some embodiments, the pharmaceutical composition of the present invention is sterile, especially for pharmaceutical use.

[0066] In some embodiments, the pharmaceutical composition of the present invention is stable upon freezing and thawing.

[0067] In some embodiments, the pharmaceutical composition of the present invention is present in vials, such as 2 mL or 20 mL type I clear glass vials.

[0068] In a preferred embodiment, the vial contains an approximate 12.5% volume overfill, or an anti-TTR antibody or antigen-binding fragment thereof with a total volume of approximately 2.25 mL or 22.5 mL.

[0069] In a preferred embodiment, the pharmaceutical composition of the present invention is a stable formulation and comprises an anti-TTR antibody as defined above. Preferably, the anti-TTR antibody comprises an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC), the immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 9, 13, 14 or 15, preferably SEQ ID NO: 9 or 15, most preferably SEQ ID NO: 15 (i.e., SEQ ID NO: 9 with the above PTM), the immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 10 at a concentration of about 25 mg / ml to about 150 mg / ml; 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); water for injection, wherein the pharmaceutical composition has a pH of about 5.3 to about 6.3.

[0070] In a generally preferred embodiment, the pharmaceutical composition of the present invention comprises 8% w / v sucrose, 0.03% w / v polysorbate 80, 20 mM histidine (e.g., L-histidine), has a pH of 5.8, and a volume of about or exactly 2.0 mL; the anti-TTR antibody or its antigen-binding fragment 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 thus comprises the human heavy chain (HC) amino acid sequence present in SEQ ID NO: 9 and the human light chain (LC) (here a κ light chain as exemplified in SEQ ID NO: 10), preferably having the above PTM, 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.

[0071] Alternatively, the pharmaceutical composition comprises 6.5% w / v sucrose, 0.03% w / v polysorbate 80, 20 mM histidine (e.g., L-histidine), has a pH of 5.8, and a volume of about or exactly 2.0 mL; and 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 wherein the antibody is a fully human IgG1m3 allotype antibody and thus comprises the human heavy chain (HC) amino acid sequence present in SEQ ID NO:9 and the human light chain (LC) (herein a κ light chain as exemplified in SEQ ID NO:10), preferably having the above PTMs, 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.

[0072] The invention also relates to a kit comprising the pharmaceutical composition of the invention and instructions for its use.

[0073] In some embodiments, the kit is used in the treatment or prevention of transthyretin-mediated amyloidosis (ATTR) in a human subject.

[0074] The invention also relates to a method of treating ATTR in a human subject, the method comprising administering the pharmaceutical composition of the invention to the human subject.

[0075] The invention also relates to a method of treating ATTR amyloid cardiomyopathy (ATTR-CM) or sporadic wild-type-ATTR-CM (e.g., the wild-type ATTR gene encoding the TTR protein that forms deposits in the heart) in a human subject, the method comprising administering the pharmaceutical composition of the invention to the human subject.

[0076] The present invention also relates to a pharmaceutical composition comprising a human anti-transthyretin (TTR) antibody or an 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; wherein the anti-TTR antibody or an antigen-binding fragment thereof is capable of binding to mutant, misfolded, misassembled, and / or aggregated TTR substances and / or fragments thereof and substantially does not recognize physiological TTR substances.

[0077] The present invention also provides a medicament comprising the pharmaceutical composition of the present invention. For example, the medicament is used in a method of treating a subject in need of treatment for ATTR, such as in treating a subject suffering from ATTR amyloid cardiomyopathy (ATTR-CM) such as sporadic WT-ATTR-CM.

[0078] The present invention also relates to a pharmaceutical container, preferably a sterile container, comprising the pharmaceutical composition of the present invention or the medicament of the present invention. For example, the container is a disposable glass vial comprising about 100 mg of an 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, such as about 100 mg / mL), optionally wherein the vial contains an approximate 10% or 12.5% volume overfill.

[0079] In a preferred embodiment, the pharmaceutical composition of the present invention is ready-to-use and is for administration to a subject in need thereof, such as by intravenous infusion, with or without dilution. Preferably, the pharmaceutical composition is a sterile, colorless to light yellow, clear to slightly opalescent liquid, substantially free of visible particles, for intravenous use by infusion after dilution.

[0080] Preferably, prior to infusion, the pharmaceutical composition is diluted in a solution containing glucose or its polymer (e.g., dextran). Thus, preferably, glucose or its polymer (e.g., dextran) is used as a diluent, and prior to infusion, the pharmaceutical composition is added to the diluent. In some embodiments, the concentration of glucose or its polymer is 5% w / v. In some embodiments, the antibody in the pharmaceutical composition is not diluted to less than 1 mg / mL.

[0081] In some embodiments, the anti-TTR antibody or antigen-binding fragment thereof is administered to a subject in an aqueous formulation at a diluted concentration of about 1 mg / mL to about 50 mg / mL (such as 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, for example 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, 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).

[0082] The present invention also relates to a method 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 one embodiment, the method comprises administering to the subject a pharmaceutical composition providing a dose of a human anti-TTR antibody of about 1 mg / kg to 60 mg / kg (e.g., about 10 mg / kg to 60 mg / kg, about 30 mg / kg to 60 mg / kg, about 30 mg / kg or about 60 mg / kg) or about 3000 mg - 7500 mg (e.g., about 4000 mg - 7000 mg, about 3000 mg - 6000 mg or about 4000 mg - 5000 mg, such as 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 a human anti-TTR antibody. In one embodiment, about 3000 mg of a human anti-TTR antibody or an antigen-binding fragment thereof is administered to a human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or an antigen-binding fragment thereof is administered to a human subject.

[0083] The present invention also relates to a method 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 one embodiment, the method comprises administering to the subject a pharmaceutical composition providing a dose of a human anti-TTR antibody of about 30 mg / kg to 60 mg / kg (e.g., about 1 mg / kg to 60 mg / kg (e.g., about 10 mg / kg to 60 mg / kg, about 30 mg / kg to 60 mg / kg, about 30 mg / kg or about 60 mg / kg) or about 3000 mg - 7500 mg (e.g., about 4000 mg - 7000 mg, about 3000 mg - 6000 mg or about 4000 mg - 5000 mg, such as 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 a human anti-TTR antibody. In one embodiment, about 3000 mg of a human anti-TTR antibody or an antigen-binding fragment thereof is administered to a human subject. In one embodiment, about 7500 mg of a human anti-TTR antibody or an antigen-binding fragment thereof is administered to a human subject.

[0084] The present invention also relates to a pharmaceutical composition of the present invention for treating ATTR (e.g., ATTR-CM or WT-ATTR-CM) in a subject in need thereof. In one embodiment, the pharmaceutical composition is for administration to a subject at a dose that provides from about 1 mg / kg to 60 mg / kg (e.g., from about 10 mg / kg to 60 mg / kg, from about 30 mg / kg to 60 mg / kg, about 30 mg / kg, or about 60 mg / kg) of a human anti-TTR antibody or from about 3000 mg - 7500 mg (e.g., from about 4000 mg - 7000 mg, from about 3000 mg - 6000 mg, or from about 4000 mg - 5000 mg, such as 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 a human anti-TTR antibody. In one embodiment, a human anti-TTR antibody or an antigen-binding fragment thereof at a dose of about 3000 mg is used to treat a human subject. In one embodiment, a human anti-TTR antibody or an antigen-binding fragment thereof at a dose of about 7500 mg is used to treat a human subject.

[0085] The present invention also relates to a pharmaceutical composition of the present invention for treating ATTR according to the present invention (e.g., ATTR-CM or WT-ATTR-CM) in a subject in need thereof. In one embodiment, the pharmaceutical composition is for administration to a subject at a dose that provides from about 1 mg / kg to 60 mg / kg (e.g., from about 10 mg / kg to 60 mg / kg, from about 30 mg / kg to 60 mg / kg, about 30 mg / kg, or about 60 mg / kg) of a human anti-TTR antibody or from about 3000 mg - 7500 mg (e.g., from about 4000 mg - 7000 mg, from about 3000 mg - 6000 mg, or from about 4000 mg - 5000 mg, such as 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 a human anti-TTR antibody. In one embodiment, a human anti-TTR antibody or an antigen-binding fragment thereof at a dose of about 3000 mg is used to treat a human subject. In one embodiment, a human anti-TTR antibody or an antigen-binding fragment thereof at a dose of about 7500 mg is used to treat a human subject.

[0086] The present invention also relates to the use of a pharmaceutical composition in the preparation of a medicament for treating or preventing ATTR in a subject. The pharmaceutical composition comprises a human anti-TTR antibody or an antigen-binding fragment thereof that can bind to mutant, misfolded, misassembled, and / or aggregated TTR substance and / or a fragment thereof, and substantially does not recognize physiological TTR substance. 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 the pharmaceutical composition of the present invention.

[0087] In some embodiments, the pharmaceutical composition comprises a human anti-TTR antibody or an 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 of sucrose, and 0.03% w / v of polysorbate 80, for the preparation of a medicament for treating or preventing ATTR in a subject. The anti-TTR antibody or an antigen-binding fragment thereof can bind to mutant, misfolded, misassembled, and / or aggregated TTR substance and / or a fragment thereof, and substantially does not recognize physiological TTR substance.

[0088] The present invention also relates to a method for obtaining the pharmaceutical composition of the present invention, which method comprises formulating an antibody in a buffer as defined above. Description of the Drawings

[0089] The drawings are included to illustrate embodiments of the present disclosure and to further understand its specific implementation.

[0090] Figure 1 is a graph depicting the size exclusion chromatography (SEC) main peak % trend of the specified buffer in the pH buffer study described in Example 1.

[0091] Figure 2 is a graph depicting the capillary isoelectric focusing (cIEF) main peak % trend of the specified buffer in the pH screening study described in Example 1.

[0092] Figure 3 is a graph depicting the decrease in SEC main peak % of six formulations (F) after incubation at 40 °C as described in Example 1.

[0093] Figure 4 is a graph depicting the decrease in SEC main peak % of the specified buffer after incubation at 40 °C for two weeks as described in Example 1.

[0094] Definition

[0095] To avoid any doubt, it should be emphasized that the use of expressions such as "in some embodiments", "in certain embodiments", "in certain cases", "in some cases", "in another embodiment", "in one embodiment", "on the other hand", "on the first hand", "on the second hand", etc., and means that any embodiment described therein is to be read in the context of each feature of those embodiments, and the present disclosure must be treated in the same manner as the combination of the features of those embodiments and aspects will be detailed in one embodiment. The same applies to any combination of embodiments and features shown in the appended claims and examples, which are also intended to be combined with the features of the corresponding embodiments disclosed in this specification, where, for the sake of consistency and brevity only, the features of the embodiments are characterized by relevance, and in fact each combination of embodiments and features (which can be understood according to the (multiple) relevance) must be regarded as literally disclosed and not as a choice among different options. In this context, those skilled in the art should understand that the embodiments and features disclosed in the examples are intended to be generalized to any anti-TTR antibody and equivalents having substantially the same properties.

[0096] As used herein, the term "about" means a value within ±10%, preferably within ±5% of the stated value. For example, "about 8%" can mean any percentage between 7.2% and 8.8%, preferably any percentage between 7.6% and 8.4%. Again, "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 the amount of antibody as mentioned herein, such as 50 mg / mL or 100 mg / mL, the term "about" refers to concentrations in the range of 45 mg / mL to 50 mg / mL (see "Acceptance Criteria" as defined in the examples) and preferably 48 mg / mL to 52 mg / mL (see, for example, Tables 4, 54, 56, 57, 60, 62, 64, 65, 67, and 68), and concentrations in the range of 90 mg / mL to 113 mg / mL, preferably 96 mg / mL to 113 mg / mL (see, for example, Tables 15, 20, 28, 34, and 46). Thus, even if the term "about" is not explicitly used, the above concentration ranges apply, for example, when referring to 50 mg / mL, concentrations in the range of 45 mg / mL to 55 mg / mL are included, and when referring to 100 mg / mL, concentrations in the range of 90 mg / mL to 113 mg / mL are included, since these ranges are within the experimental deviations shown in the above tables.

[0097] In the context of the present invention, the term "and / or" shall be understood to mean that all members of the group connected by the term "and / or" are cumulatively disclosed in any combination, alternately combined with each other, and combined with each other in each case. For the expression "A, B and / or C", this means that the following disclosures should be understood as follows: 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).

[0098] In the context of the formulations / drug compositions of the present invention, the phrases "substantially free of NaCl" and "essentially free of NaCl" mean that the drug 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 a low level that does not affect the performance, stability, safety, or efficacy of the formulation. Additionally, the phrase can also refer to a formulation / drug composition in which NaCl has not been intentionally added, but which may contain Na 2+ or Cl - .

[0099] In the context of the formulations / drug compositions of the present invention, the phrases "substantially free of" and "essentially free of" mean that the drug composition / formulation does not contain the substance mentioned or contains only trace amounts that are considered negligible for the intended use of the product, i.e., the substance is present at a low level that does not affect the performance, stability, safety, or efficacy of the formulation.

[0100] As used herein, the term "binding potency" refers to a characteristic corresponding to a quantitative measure of biological activity (e.g., TTR binding). Binding potency assays (e.g., ELISA assays) can be used to measure the ability of the anti-TTR antibodies or antigen-binding fragments thereof disclosed herein to elicit a specific response in a disease-related system (e.g., a subject with ATTR-CM such as WT-ATTR-CM). The activity measured in the assay is a surrogate for the expected biological effect and can be used to evaluate the maintenance of that effect over time (e.g., after storage).

[0101] As used herein, the expressions "capable of binding" and "binds to" refer to the ability of an antibody or antigen-binding fragment thereof to bind to, for example, aggregated TTR under the experimental conditions exemplified in the Examples (e.g., in an ELISA assay). As used herein, the term "drug composition" refers to a mixture containing a therapeutic agent (e.g., an anti-TTR antibody as described herein) optionally combined with one or more pharmaceutically acceptable excipients, diluents, and / or carriers. The drug composition is formulated, for example, for administration to a subject, such as a mammal, e.g., a human, to prevent, treat, or control a specific disease or disorder that affects or may affect the subject (e.g., ATTR-CM, such as WT-ATTR-CM).

[0102] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without excessive toxicity, irritation, allergic response, and other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0103] As used herein, the term "between" includes the endpoints.

[0104] As used herein, according to the European Pharmacopoeia, room temperature (RT) is defined as being between 15°C and 25°C. The "percent sequence identity (%)" relative to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in the candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity. The alignment for determining the 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, e.g., using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning the sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. For example, the sequence comparison computer program BLAST can be used to generate percent sequence identity values. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A relative to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be stated as a given nucleic acid or amino acid sequence A has a certain percent sequence identity relative to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows:

[0105] 100 multiplied by (fraction X / Y)

[0106] where X is the number of nucleotides or amino acids scored as identical matches by the program in the alignment of sequences A and B (e.g., BLAST), and where Y is the total number of nucleic acids in B. It should be understood that in the case where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A. Detailed Description

[0107] The present disclosure relates to a pharmaceutical composition containing an anti-transthyretin (TTR) antibody or an antigen-binding fragment thereof, which can be used in various therapeutic and prophylactic methods described herein. The pharmaceutical composition contains an antibody or an antigen-binding fragment thereof, which can bind to mutant, misfolded, misassembled, and / or aggregated TTR substances and / or fragments thereof and substantially does not recognize physiological TTR substances.

[0108] The pharmaceutical composition contains a combination of specific carriers and excipients, which endows the composition with surprising beneficial properties, including the ability to formulate a drug (an anti-TTR antibody and its antigen-binding fragments) at a high concentration (e.g., about 150 mg / mL), enhance the stability of the composition (e.g., extended shelf life, about 4 weeks especially at high temperature (e.g., at about 40 °C) and 18 months at low temperature (e.g., at about 5 °C)), reduce the aggregation of the drug (e.g., ≤ 400 particles / mL with a size ≥ 10 μM), and improve the viscosity parameters of the composition (e.g., about 16 cP). The components of the pharmaceutical composition are described in more detail below.

[0109] Antibodies and their antigen-binding fragments

[0110] The pharmaceutical composition described herein comprises an antibody or an antigen-binding fragment thereof that is capable of binding to aggregated human wild-type transthyretin (wtATTR) and preferably does not bind to monomers and dimers of human native TTR, such as a human anti-TTR antibody. Preferably, the antibody is also capable of binding to mutant TTR aggregates.

[0111] 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. The CDR sequences are defined by 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).

[0112] 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).

[0113] Table 1. Anti-TTR antibody sequences

[0114]

[0115]

[0116]

[0117] CDR = Complementary Determining Region; VH = Variable Region of Heavy Chain; VL = Variable Region of Light Chain; HC = Heavy Chain; LC = Light Chain. The N-terminal glutamine is absent and / or has been modified to pyroglutamic acid, which is also known as N-terminal cyclization. Preferably, the N-terminal glutamine has been modified to pyroglutamic acid

[0118] An anti-TTR antibody or an antigen-binding fragment thereof may comprise one or more CDR sequences, the CDR sequences comprising 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 sequences 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 an antigen-binding fragment thereof may comprise one or more CDR sequences, the CDR sequences having amino acid sequences with 1, 2 or 3 mismatches relative 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 / or SEQ ID NO:6. In one specific example, an anti-TTR antibody or an 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 an antigen-binding fragment thereof may have a VH region that comprises 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 one specific example, an anti-TTR antibody or an antigen-binding fragment thereof has a VH region that comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7. An anti-TTR antibody or an antigen-binding fragment thereof may have a VL region that comprises 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 one specific example, an anti-TTR antibody or an antigen-binding fragment thereof has a VL region that comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:8.

[0119] The anti-TTR antibody or its antigen-binding fragment may have a VH region that comprises 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 specific example, the anti-TTR antibody or its antigen-binding fragment has a VH region that comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:11.

[0120] The anti-TTR antibody or its antigen-binding fragment may have a VL region that comprises 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 specific example, the anti-TTR antibody or its antigen-binding fragment has a VL region that comprises an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:12.

[0121] Alternatively, the anti-TTR antibody is the anti-TTR antibody described in U.S. Patent 10,344,080 or 11,180,545 (each of the patents is incorporated herein by reference in its entirety). In some embodiments, the anti-TTR antibody is the anti-TTR antibody described in U.S. Publication US2022-0144928 (the publication is incorporated herein by reference in its entirety).

[0122] In addition, anti-TTR antibodies and antigen-binding fragments thereof are within the scope of the present disclosure, in which specific amino acids have been substituted, deleted, or added. These modifications do not significantly affect the biological properties of the anti-TTR antibody, such as binding activity. For example, the antibody may have amino acid substitutions in the framework region (FR) to improve binding to the antigen. As another example, multiple 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 on how to make phenotypically silent amino acid substitutions is provided in, for example, 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, N.Y., 1989), Pearson (Methods Mol. Biol. 243:307-31, 1994), and Gonnet et al. (Science 256:1443-45, 1992); each of these references is incorporated herein by reference.

[0123] The variant antibody or its antigen-binding fragment is functionally active and may have, for example, a number of substituted or deleted residues of less than about 30%, about 25%, about 20%, about 15%, about 10%, about 5% or about 1% of the amino acids while retaining substantially the same immunological properties, including but not limited to binding to TTR, as described herein, i.e., an equivalent antibody having substantially the same TTR-binding properties as the exemplary antibody, which has been described in U.S. Patent Nos. 10,344,080 B2 and 11,180,545 B2 (corresponding to International Application WO 2015 / 092077 A1, in which the antibody is named NI-301.37F1; see also ibid.), to comprise a heavy chain variable region having the amino acid sequence of SEQ ID NO:7 and a light chain variable region having the amino acids of SEQ ID NO:8. As mentioned above, the antibody is characterized in U.S. Patent Nos. 10,344,080 B2 and 11,180,545 B2 and in Michalon et al., Nat. Commun. 12 (2021), 3142 (named antibody NI301A) by: (i) selectively binding to disease-associated ATTR aggregates with high affinity, (ii) binding to misfolded / aggregated wild-type TTR and variant TTRs associated with sporadic and inherited diseases, respectively, and binding to ATTR deposits in heart tissue obtained at autopsy from ATTR-CM patients, but substantially not binding to native TTR monomers, and importantly, being able to remove ATTR fibrils by macrophage-mediated phagocytosis. The latter property can be easily tested as described in International Application WO 2020 / 094883 A1; see also Michalon et al. (2021), ibid.

[0124] The antibody or its antigen-binding fragment may also comprise variants, including, for example, humanized or chimeric antibodies or their antigen-binding fragments, analogs of antibodies, orthologs, homologs and derivatives, which exhibit biological activity, such as binding to an antigen such as TTR. The antibody may contain one or more amino acid analogs (including, for example, non-naturally occurring amino acids, amino acids that are only naturally present in unrelated biological systems, modified amino acids from mammalian systems, etc.), antibodies having substituted bonds, and other modifications known in the art.

[0125] In some embodiments, the anti-TTR antibody fragment is selected from the group consisting of diabodies, Fab, Fab’-SH, Fv, scFv and (Fab’)2 fragments.

[0126] In certain embodiments, the anti-TTR antibody or an antigen-binding fragment thereof is a monoclonal antibody (mAb). In some cases, the anti-TTR antibody or an antigen-binding fragment thereof is an IgG antibody. In some cases, the anti-TTR antibody or an antigen-binding fragment thereof is a human IgG1 antibody. In one embodiment, the human anti-TTR antibody or an 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 an antigen-binding fragment thereof has a VH region and a VL region, the VH region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:7, and the VL region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:8.

[0127] In this context, those skilled in the art know that effector functions and strengths can depend especially on the IgG class or isotype, and that IgG2 and IgG4 have only attenuated effector functions compared to IgG1 or IgG3. Thus, in one embodiment, the anti-TTR antibodies described herein can be of the IgG1 or IgG3 class or isotype, such as IgG1. Of course, in addition to using natural IgG immunoglobulins, the corresponding effector functions can also be genetically engineered; see, for example, Saunders KO (2019) Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life. Front. Immunol. 10:1296. Doi:10.3389 / fimmu.2019.01296.

[0128] The five main classes of immunoglobulins are IgG, IgM, IgA, IgD, and IgE. These classes are distinguished by the type of heavy chain present in the molecule. IgG molecules have a heavy chain called the γ chain; IgM has a μ chain; IgA has an α chain; IgE has an ε chain; and IgD has a δ chain; see reviews such as Schroeder et al., J. Allergy Clin. Immunol. 125 (2010), S41-S52. In addition, there are different subclasses, where IgA is further divided into subclasses IgA1 and IgA2, and where IgG is further divided into subclasses IgG1, IgG2, IgG3, and IgG4. In addition, there are two types of light chains, κ and λ.

[0129] In principle, the antibodies used according to the present invention can be of any class and subclass, respectively, and can comprise any kind of light chain, provided that the antibody binds to misfolded and preferably aggregated forms of TTR, and preferably provided that the binding specificity for TTR as shown in the examples of WO 2015 / 092077 A1 remains unaffected in kind for the antibody NI-301.37F1, and provided that no adverse effects occur when the antibody is administered to a subject, wherein the adverse effects can be determined as described in Example 1. However, preferably intact IgG antibodies are used, wherein the antibody comprises constant domains. Thus, in one embodiment, the immunoglobulin heavy chain and / or light chain constant domains present in the antibodies used according to the present invention are of the IgG type, IgM type, IgA type, IgD type or IgE type, preferably of the IgG type. In one embodiment, the immunoglobulin heavy chain and / or light chain constant domains present in the antibodies used according to the present invention are of the IgA1, IgA1, IgG1, IgG2, IgG3 or IgG4 subclass, preferably of the IgG1, IgG2, IgG3 or IgG4 subclass, and most preferably of the IgG1 subclass.

[0130] The recombinant expression of intact human IgG1 antibodies with human or murine constant domains can be carried out substantially as described in the examples of WO 2015 / 092077 A1. Preferably, the antibody is a monoclonal antibody or derived from a monoclonal antibody.

[0131] Not only are there the four above-mentioned IgG subclasses, but the human heavy and light chain genes also exhibit extensive structural polymorphisms and are inherited tightly linked as haplotypes. Allotypic variants can be immunogenic and elicit antibody responses due to alloimmunization. Thus, switching allotypes can be of particular interest to provide non-immunogenic antibody therapeutics. To date, extensive allotypes (polymorphisms) are known, but it is important the serologically defined allotypes. The allotypes of IgG proteins are defined by the expression of unique epitopes recognized by unique serum reagents. The allotypes expressed on the constant region of the IgG heavy chain are named Gm (genetic marker) together with the subclass such as G1m and the allotype number (or letter) such as 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 Irani et al., Molecular Immunology 67 (2015), 171-182 Figure 1In A, the content of the literature is incorporated herein by reference. Thus, in one embodiment, the antibodies used in accordance with the present invention are any of the following allotypes, but are not limited to: 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 preferably G1m2, G1m3, or G1m17, and most preferably G1m3.

[0132] As described above, the antibody NI006 / ALXN2220 is a fully human IgG1m3 allotype antibody and consists of two identical heavy chains of the IgG1 subclass and the IgG1m3 allotype. Additionally, as mentioned above, the original human antibody NI-301.37F1 is of the κ type, and thus NI006 / ALXN2220 consists of two identical light chains of the κ subclass. The sequences of the variable heavy (VH) and variable light (VL) chains of NI006 / ALXN2220 are shown in SEQ ID NO: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 for its heavy chain constant region; see Jefferis and Lefrance (2009), supra. Thus, in one embodiment, the antibody present in the pharmaceutical formulation of the present invention is characterized by two heavy chains and two light chains, wherein each heavy chain (HC) contains the amino acid sequence shown in SEQ ID NO:9 and each light chain (LC) contains the amino acid sequence shown 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-chain and inter-chain disulfide bonds, and the positions of the disulfide bonds that have been identified by Lys-C and trypsin digestion and subsequent LC-MS (see Example 2) are as follows:

[0133] LC: C23 - LC: C88

[0134] LC: C134 - LC: C194

[0135] LC: C214 - HC: C223

[0136] HC: C22 - HC: C97

[0137] HC: C147 - HC: C203

[0138] HC1: 229 - HC2: 229 and HC1: 232 - HC2: 232

[0139] HC: C264 - HC: C324

[0140] HC: C370 - HC: C428.

[0141] (The amino acid numbering corresponds to the heavy chain sequence shown in SEQ ID NO:9)

[0142] Thus, the antibodies present in the pharmaceutical compositions of the present invention may be characterized by comprising at least 8 disulfide bridges, preferably at the positions identified above.

[0143] In addition, each heavy chain of the antibody NI006 / ALXN2220 contains a single N-linked glycosylation site at Asn300. The N-linked glycosylation structure is mainly a fucosylated complex biantennary glycan that has 0 galactose residues (G0F) (about 49%) or has 1 galactose residue (G1F) (about 25%). The detailed glycosylation characteristics are shown in Example 2. Glycosylation plays a crucial 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 valuable information on the safety and efficacy of biotherapeutics.

[0144] Thus, in a preferred embodiment, the antibodies present in the pharmaceutical compositions of the present invention are IgG antibodies and have N-glycosylated heavy chains, preferably where the N-linked glycosylation site is Asn300, preferably, if expressed, for example, in CHO cells, where the antibody comprises an N-linked glycosylation structure that is mainly a glycan having 0 galactose residues (G0F) (about 49%) or having 1 galactose residue (G1F) (about 25%). Most preferably, the antibody has the glycosylation characteristics shown in Example 3.

[0145] In addition, one or several amino acids at the amino or carboxyl terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain (if present), may be deleted or derivatized in part or all of the molecule.

[0146] Thus, in one embodiment, the antibodies present in the pharmaceutical compositions of the present invention have heavy chains that do not contain C-terminal lysine. For example, in such embodiments, the C-terminal lysine contained in SEQ ID NO:9 is deleted. The sequence of such heavy chains is shown in SEQ ID NO:13.

[0147] In addition or alternatively, the antibody has a heavy chain in which the N-terminal glutamine is derivatized, preferably replaced by pyroglutamic acid. This formation of pyroglutamic acid is also known as N-terminal cyclization. The sequence of such heavy chains is shown in SEQ ID NO:14 or SEQ ID NO:15.

[0148] Most preferably, the antibody has a heavy chain that does not contain a C-terminal lysine, i.e., the C-terminal lysine has undergone C-terminal lysine cleavage, and the N-terminal glutamine has been replaced by pyroglutamic acid, i.e., it has undergone N-terminal glutamine acylation cyclization (see SEQ ID NO: 15), and it is N-glycosylated.

[0149] The amino acid sequences of the heavy and light chains are shown below:

[0150] QLQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0151] (SEQ ID NO: 9, NI006 / ALXN2220, heavy chain amino acid sequence, where the amino acids of the constant region are underlined, and where the C-terminal lysine (K) is optional and / or the N-terminal glutamine (Q) undergoes intramolecular cyclization, resulting in the formation of pyroglutamic acid)

[0152] DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0153] (SEQ ID NO: 10, NI006 / ALXN2220, light chain amino acid sequence, where the amino acids of the constant region are underlined).

[0154] Furthermore, the theoretical molecular weight of the antibody NI006 / ALXN2220 is 144.2 kDa, and the weights determined by mass spectrometry (MS) are 144.2 kDa (deglycosylated) and between 147.0 and 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.

[0155] Formulation

[0156] The present disclosure has a pharmaceutical composition containing the above-mentioned anti-TTR antibody or its antigen-binding fragment. The pharmaceutical composition of the present invention can be formulated as described below. For example, the pharmaceutical composition containing the anti-TTR antibody can be formulated to contain sucrose, polysorbate (preferably polysorbate 80) and / or a polar excipient (e.g., histidine). In addition, the pharmaceutical composition containing the anti-TTR antibody can be formulated at the desired pH described herein (e.g., pH 5.8). The pharmaceutical composition containing the anti-TTR antibody may also contain a pharmaceutically acceptable excipient or diluent as described herein.

[0157] For example, the pharmaceutical composition can include a human anti-TTR antibody or antigen-binding fragment thereof (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 and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and the 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 volume of 2.0 mL, a histidine buffer at a pH of about 5.8, 6.5% w / v sucrose, and 0.03% w / v polysorbate 80.

[0158] As another example, the pharmaceutical composition can include a human anti-TTR antibody or antigen-binding fragment thereof (e.g., an anti-TTR antibody or antigen-binding fragment thereof having a VH region and a VL region, the VH region comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:7, the 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 and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:9, the light chain comprising an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO:10) all at a concentration of about 50 mg / mL in a volume of 2.0 mL, a histidine buffer at a pH of about 5.8, 8.0% w / v sucrose, and 0.03% w / v polysorbate 80.

[0159] As shown in Example 3, about 99% to 100% of the antibodies present in the pharmaceutical formulation have N-terminal pyroglutamic acid in the heavy chain, and about 96% of the antibodies have a deletion of the C-terminal lysine. Thus, in one embodiment, about 99% of the antibodies in the formulation of the present invention have a heavy chain in which the N-terminal pyroglutamic acid is modified from N-terminal glutamine, and / or about 96% of the antibodies have a deletion of the C-terminal lysine.

[0160] Thus, the pharmaceutical composition may comprise a human anti-TTR antibody or an antigen-binding fragment thereof (e.g., an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, the VH region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 11, the VL region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 8; and an anti-TTR antibody having a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 13, 14 or 15, preferably SEQ ID NO: 15, and the light chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 10) at a concentration of about 50 mg / mL in a 2.0 mL volume, a histidine buffer at a pH of about 5.8, 6.5% w / v sucrose, and 0.03% w / v polysorbate 80.

[0161] Alternatively, the pharmaceutical composition may comprise a human anti-TTR antibody or an antigen-binding fragment thereof (e.g., an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, the VH region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 11, the VL region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 8; and an anti-TTR antibody having a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 13, 14 or 15, preferably SEQ ID NO: 15, and the light chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 10) at a concentration of about 50 mg / mL in a total 2.0 mL volume, a histidine buffer at a pH of about 5.8, 8.0% w / v sucrose, and 0.03% w / v polysorbate 80.

[0162] In addition, but to a lesser extent and preferably in negligible amounts, some antibody species may be present in the antibody composition being analyzed that may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation, and isomerization. PTMs identified as present in NI006 / ALXN2220 are mentioned in Example 3. Specifically, after the aforementioned C-terminal lysine cleavage and N-terminal cyclization, the antibody may exhibit methionine (M) oxidation, e.g., at HC position 255; asparagine (N) deamidation, e.g., at HC positions 318 and / or at HC position 387; asparagine (N) succinimide formation, e.g., at HC position 318; and / or amidation of C-terminal proline (P) after C-terminal lysine and glycine deletion.

[0163] Antibody concentration

[0164] Any anti-TTR antibody or antigen-binding fragment thereof described herein (e.g., an anti-TTR antibody comprising 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, and an anti-TTR antibody comprising a heavy chain having the amino acid sequence of any one of SEQ ID NOs: 13 to 15 or SEQ ID NO: 9 and a light chain having the amino acid sequence of SEQ ID NO: 10) can be formulated into a pharmaceutical composition described herein at a concentration of about 1 mg / mL to about 500 mg / mL.

[0165] The pharmaceutical composition may comprise from about 1 mg / mL to about 400 mg / mL (such as from about 1 mg / mL to about 100 mg / mL, such as 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, such as from about 100 mg / mL to about 200 mg / mL, such as 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, such as from about 200 mg / mL to about 300 mg / mL, such as 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, such as from about 300 mg / mL to about 400 mg / mL, such as 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) of the anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise from about 1 mg / mL to about 300 mg / mL of the anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise from about 1 mg / mL to about 200 mg / mL of the anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise from about 1 mg / mL to about 100 mg / mL (such as from about 1 mg / mL to about 20 mg / mL, such as about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL or about 20 mg / mL, such as from about 20 mg / mL to about 40 mg / mL, such as about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL or about 40 mg / mL, from about 40 mg / mL to about 60 mg / mL, such as 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, such as from about 60 mg / mL to about 80 mg / mL, such as about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL or about 80 mg / mL, such as from about 80 mg / mL to about 100 mg / mL, such as about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL or about 100 mg / mL) of the anti-TTR antibody or antigen-binding fragment thereof described herein.The pharmaceutical composition may comprise from about 30 mg / mL to about 70 mg / mL of the anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise from 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 the anti-TTR antibody or antigen-binding fragment thereof described herein.

[0166] 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 the anti-TTR antibody or antigen-binding fragment thereof described herein. The pharmaceutical composition may comprise about 50 mg / mL of the anti-TTR antibody or antigen-binding fragment thereof described herein.

[0167] Sucrose

[0168] The pharmaceutical composition may further comprise sucrose, for example, in an amount of from about 6% to about 9%, from about 6% to about 7% or from about 7.5% to about 8.5% weight / unit volume (w / v) (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5% or 9% w / v sucrose). For example, the pharmaceutical composition may comprise 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. Specifically, the pharmaceutical composition comprises about 6.5% w / v sucrose or about 8% w / v sucrose.

[0169] Polysorbate

[0170] The pharmaceutical composition may also comprise a polysorbate, such as polysorbate 20 or polysorbate 80, preferably polysorbate 80 (PS80), for example in an amount of from about 0.001% to about 0.1% w / v (for example, about 0.001%, 0.005%, 0.01%, 0.05% or 0.1% w / v PS(80)). For example, the pharmaceutical composition may comprise 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. Specifically, the pharmaceutical composition comprises about 0.03% w / v PS(80).

[0171] Polar excipient / buffer system

[0172] The pharmaceutical composition may further comprise a polar excipient. The polar excipient may be or comprise, 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 contain buffers and are known in the art, and include, for example, citrate buffers, phosphate buffers, acetate buffers, histidine buffers and combinations thereof. Suitable buffers can be selected in particular using the guidance provided above. The preferred buffer used according to the present invention is a histidine buffer. The pharmaceutical composition may comprise a polar excipient (buffer) (e.g., histidine) in an amount of, for example, from 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 drug may comprise a polar excipient (e.g., histidine) at 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 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 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. Specifically, the pharmaceutical composition comprises a polar excipient (e.g., histidine) at about 20 mM.

[0173] pH

[0174] 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. Specifically, the pharmaceutical composition has a pH of about 5.8.

[0175] Excipients and diluents

[0176] The pharmaceutical composition may comprise a pharmaceutically acceptable excipient (e.g., buffer, carrier, stabilizer or preservative) or diluent (e.g., saline and aqueous buffer solutions). Pharmaceutically acceptable buffers include, for example, bacteriostatic water for injection (BWFI), phosphate buffered saline (PBS), Ringer's solution and dextrose solution.

[0177] Volume

[0178] The pharmaceutical composition may be provided in a volume of from about 1 mL to about 100 mL, from about 1 mL to about 50 mL, from about 5 mL to about 25 mL, from about 20 mL to about 25 mL or from about 1 mL to about 10 mL (e.g., from about 1 mL to about 80 mL, from about 1 mL to about 70 mL, from about 1 mL to about 60 mL, from about 1 mL to about 50 mL, from about 1 mL to about 40 mL, from about 1 mL to about 30 mL, from about 1 mL to about 20 mL, from about 1 mL to about 10 mL, from about 5 mL to about 20 mL, from about 5 mL to about 15 mL, from about 5 mL to about 10 mL, from about 10 mL to about 20 mL, from about 15 mL to about 20 mL, from about 20 mL to about 22.5 mL, from about 20 mL to about 25 mL, from about 1 mL to about 9 mL, from about 1 mL to about 8 mL, from about 1 mL to about 7 mL, from about 1 mL to about 6 mL, from about 1 mL to about 5 mL, from about 1 mL to about 4 mL, from about 1 mL to about 3 mL, from about 1 mL to about 2.25 mL, from about 1 mL to about 2 mL or from about 2 mL to about 2.25 mL) (e.g., in a vial or other container as described herein). For example, the pharmaceutical composition may be present in a volume of from about 1 mL to about 2.25 mL (e.g., from about 1 mL to about 2.2 mL, from about 1 mL to about 2 mL, from about 1 mL to about 1.8 mL, from about 1 mL to about 1.6 mL, from about 1 mL to about 1.4 mL, from about 1 mL to about 1.2 mL, from about 1.5 mL to about 1.25 mL, from about 1.5 mL to about 2 mL, from about 1.9 mL to about 1.2 mL, from about 2.1 mL to about 2.25 mL) or from about 1 mL to about 100 mL (e.g., about 1 mL, about 1.8 mL, about 1.9 mL, about 2 mL, about 2.1 mL, about 2.2 mL, 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).

[0179] In one specific 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.

[0180] Additional formulation

[0181] A pharmaceutical composition can be prepared such that it is not reconstituted from the lyophilized anti-TTR antibody and / or is not further lyophilized. Additionally, a pharmaceutical composition can be prepared such that it is substantially free of sodium chloride and / or substantially free of poloxamer. The pharmaceutical composition can also be prepared as a sterile composition.

[0182] In the most preferred embodiment of the present invention, the pharmaceutical composition comprises or consists of the following: 50 mg / mL antibody (such as 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 at pH 5.8.

[0183] Characteristic

[0184] The present disclosure features an invention of a pharmaceutical composition having improved characteristics (e.g., stability, solubility, storage, etc.), as described herein (e.g., see Examples 1 and 2).

[0185] The pharmaceutical composition can be characterized by having a storage life of 24 months at 2 to 8 °C, preferably in the dark.

[0186] The pharmaceutical composition can be characterized by any combination of one, two, three, or all four of the following stability criteria (e.g., (a) to (d)): (a) the main peak decrease under heat stress conditions at 40 °C for 4 weeks and / or at about 25 °C for 12 weeks is less than 1% by weight of the antibody, as measured by size exclusion chromatography (SEC)-HPLC analysis; (b) the pharmaceutical composition shows that the content of acidic species of the anti-TTR antibody under heat stress conditions at about 40 °C for 2 weeks is less than 42.5%, as measured by capillary isoelectric focusing (cIEF); (c) after 3 cycles of freeze-thaw (about -70 °C to room temperature, e.g., 20 °C - 26 °C, such as 25 °C), the pharmaceutical composition shows no significant change in the content of acidic species of the anti-TTR antibody, as measured by capillary isoelectric focusing (cIEF); and / or (d) the anti-TTR antibody retains at least 80% of its binding efficacy to TTR protein after storage at 40 °C for 4 weeks and / or at least 70% of its binding efficacy to TTR protein after storage at 25 °C for 12 weeks, e.g., as measured by ELISA and relative to a control (e.g., not stored for a long time).

[0187] For example, the pharmaceutical composition shows a main peak ≥ 50.0%, an acidic peak ≤ 40.0%, and a basic peak ≤ 15.0% as measured by, for example, cIEF, a main peak (monomer) ≥ 95.0% and high molecular weight substances (HMWS) ≤ 5.0% as measured by, for example, size exclusion chromatography (SEC)-HPLC analysis, a pH of 5.8 ± 0.5, an osmolality ≥ 240 mOsm / Kg, and an antibody concentration of 50 ± 5.0 mg / mL.

[0188] Furthermore, the pharmaceutical composition of the present invention may be characterized by any combination of one, two, three, or all four stability criteria (i) to (iv): (i) a decrease in the main peak (indicating monomer content) of 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 of about 40 °C for 1 month or about 25 °C for 6 months or during long-term storage at about 5 °C for 18 months; (ii) the pharmaceutical composition shows a content of acidic substances of the anti-TTR antibody or its antigen-binding fragment of less than or approximately equal to about 40% under heat stress conditions of about 40 °C for 2 weeks or about 25 °C for 3 months as measured by cIEF; (iii) the pharmaceutical composition shows a content of acidic substances of the anti-TTR antibody or its antigen-binding fragment of less than 40%, preferably less than 35% as measured by cIEF under long-term storage conditions of about 5 °C for 12 months or 18 months; and / or (iv) the anti-TTR antibody or its antigen-binding fragment retains at least 80%, preferably at least 90% of its binding efficacy to the TTR protein after storage at about 25 °C for 6 months or at about 5 °C for 12 months or 18 months as measured by ELISA and relative to a control (e.g., not stored for a long time).

[0189] Preferably, the pharmaceutical composition shows any one or all of the characteristics shown in Tables 56 to 69 of the Examples.

[0190] Specifically, the pharmaceutical composition has an osmolality of ≥ 240 mOsm / Kg and contains 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).

[0191] The pharmaceutical composition is stable upon freezing and thawing. As used herein, the terms "stable" or "stability" as used in the context of the pharmaceutical compositions described herein refer to the maintenance of the physical and functional characteristics of the composition over time. For example, a stable composition can be described as one that maintains its appearance (e.g., color, opalescence, number of visible particles, and / or number of sub-visible particles), pH, antibody concentration, and / or osmolality after long-term storage (e.g., for 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 (e.g., 1, 2, 3, 4, 5, or more) freeze-thaw cycles. Additionally, a stable composition can be described as one that maintains its functionality (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 (e.g., 1, 2, 3, 4, 5, or more) freeze-thaw cycles.

[0192] The pharmaceutical composition of the present invention has been shown to be stable for at least 1 month at 40 ± 2°C and 75 ± 5% RH (stress stability study); stable for at least 6 months at 25 ± 2°C / 60 ± 5% RH (accelerated stability study); and / or stable for at least 12 to 18 months at 5 ± 3°C (long-term stability study). Additionally, the extinction coefficient of the pharmaceutical composition has been measured to be 1.438 (mg / mL) -1cm-1 , and the pharmaceutical formulation has been shown to be a sterile, colorless to light yellow, clear to slightly opalescent solution, substantially free of visible particles, with a pH of 5.8.

[0193] Treatment method

[0194] A pharmaceutical composition comprising an anti-TTR antibody or an antigen-binding fragment thereof (e.g., an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, the VH region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7 (e.g., having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with SEQ ID NO: 7, or 100% sequence identity with SEQ ID NO: 11, and also having, for example, the CDR sequences shown in SEQ ID NOs: 1-3), the VL region comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8 (e.g., having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, and having, for example, the CDR sequences shown in SEQ ID NOs: 4-6); and an anti-TTR antibody having a heavy chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 9, preferably comprising the PTMs mentioned above, i.e., an anti-TTR antibody having a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 13, 14 or 15, preferably SEQ ID NO: 15, and the light chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO: 10) can be administered to a subject (e.g., a human) to treat, prevent or control a disease or disorder as described herein. For example, the pharmaceutical composition described herein can be used in a method for treating or preventing transthyretin-mediated amyloidosis (ATTR). Additionally, the pharmaceutical composition described herein can be used in a method for treating or preventing ATTR amyloid cardiomyopathy (ATTR-CM, such as WT-ATTR-CM).For example, a pharmaceutical composition containing a human anti-TTR antibody or an antigen-binding fragment thereof (e.g., an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, the VH region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:7 or 100% sequence identity with SEQ ID NO:11, the VL region comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:8; and an anti-TTR antibody having a heavy chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:9, preferably comprising the PTMs mentioned above, i.e., an anti-TTR antibody having a heavy chain and a light chain, the heavy chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:13, 14 or 15, preferably SEQ ID NO:15, and the light chain comprising an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:10), 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 can be administered to treat ATTR or ATTR-CM, such as WT-ATTR-CM.

[0195] A pharmaceutical composition containing an anti-TTR antibody can be administered to a human subject to treat, prevent or control transthyretin-mediated amyloidosis (ATTR), including ATTR amyloid cardiomyopathy (ATTR-CM, such as WT-ATTR-CM). The pharmaceutical composition can be administered by intravenous injection or infusion to provide a dose of 30 mg / kg 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, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, 3000 mg, 3000 mg, 3100 mg, 3200 mg, 3300 mg, 3400 mg, 3500 mg, 3600 mg, 3700 mg, 3800 mg, 3900 mg, 4000 mg, 4100 mg, 4200 mg, 4300 mg, 4400 mg, 4500 mg, 4600 mg, 4700 mg, 4800 mg, 4900 mg, 5000 mg, 5100 mg, 5200 mg, 5300 mg, 5400 mg, 5500 mg, 5600 mg, 5700 mg, 5800 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) of human anti-TTR antibody to the subject. For example, a dose of 3000 mg or a dose of 7500 mg and doses in between can be used to treat a human subject.

[0196] The compositions and methods provided herein can be used to treat subjects suffering from: ATTR, ATTR-CM, ATTR polyneuropathy (ATTR-PN), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, leptomeningeal / 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 known pathogenic TTR mutations (e.g., mutations causing amyloidosis). The subject may have a negative genetic test for sporadic WT-ATTR-CM and TTR mutations.

[0197] The pharmaceutical compositions of the disclosure can be formulated for administration by a variety of methods known in the art. The administration can be, for example, intravenous or subcutaneous. Intravenous delivery by continuous infusion is one method for administering the pharmaceutical compositions disclosed herein.

[0198] The pharmaceutical composition can be ready-to-use for administration to a subject in need, preferably via intravenous infusion. The pharmaceutical composition can be diluted with glucose or a polymer thereof prior to infusion, preferably wherein the polymer is dextran. The concentration of glucose or its polymer can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w / v.

[0199] In some cases, the pharmaceutical composition is administered according to the dosing regimens described in ClinicalTrials.gov ID NCT04360434 and Garcia-Pavia et al. (2023) and European Patent Applications EP 22 207 651.5 and EP 23 020 175.8; U.S. Patent Application 63 / 383807; and International Application PCT / EP2023 / 081809 filed on November 15, 2023, which international application claims the priority of EP 22 207651.5, EP 23 020 175.8, and US 63 / 383807 (incorporated herein by reference). Thus, in a preferred embodiment, the pharmaceutical composition of the invention, particularly the most preferred composition of NI006 / ALXN2220 mentioned above, is administered approximately every 28 days.

[0200] Product

[0201] The present disclosure also has an article (e.g., a kit) that contains materials useful for treating or preventing transthyretin-mediated amyloidosis (ATTR) in a human subject. The article includes 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 a label or package insert located on or associated with 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 sized 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL or 25 mL and are capable of accommodating a drug composition that is overfilled by about 10% to about 15% (e.g., 10% or 12.5%) in volume. The container can include a volume of the drug composition described herein, such as a volume of 0.5 mL to 10 mL, 2 mL to 2.25 mL, 10 mL to 20 mL, 15 mL to 25 mL, 20 mL to 22.5 mL, for example 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 drug composition (i.e., the drug product described herein) is provided as a concentrate of a solution for infusion, which is presented as a sterile, colorless to light yellow, clear to slightly milky white liquid, substantially free of visible particles, and is provided in a 2 mL (2R) glass vial with an aluminum flip-top above a 13 mm rubber stopper. The product is preferably diluted in sterile glucose prior to administration, which is a commercial product and, in one embodiment, is not accompanied by the drug product.

[0202] The pharmaceutical composition may contain, for example, a human anti-TTR antibody or an antigen-binding fragment thereof 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), such as an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, wherein the VH region contains an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:11, and the VL region contains an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:8; and an anti-TTR antibody having a heavy chain, wherein the heavy chain contains an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:9, preferably containing the PTM mentioned above, i.e., an anti-TTR antibody having a heavy chain and a light chain, wherein the heavy chain contains an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:13, 14 or 15, preferably SEQ ID NO:15, and the light chain contains an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:10), and having, for example, a histidine buffer at a pH of about 5.8, 6.5% or 8.0% w / v of sucrose, and 0.03% w / v of polysorbate 80.The pharmaceutical composition of the article may contain, for example, an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region in a total amount of about 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, such as 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), wherein the VH region contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:7 (e.g., having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:11, and having, for example, the CDR sequences shown in SEQ ID NO:1-3), the VL region contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:8 (e.g., having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:8, and having, for example, the CDR sequences shown in SEQ ID NO:4-6), and an anti-TTR antibody having a heavy chain with the amino acid sequence of SEQ ID NO:9, preferably containing the PTM mentioned above, i.e., an anti-TTR antibody having a heavy chain and a light chain, the heavy chain containing an amino acid sequence having 100% sequence identity with the amino acid sequence of SEQ ID NO:13, 14 or 15, preferably SEQ ID NO:15, and the light chain having the amino acid sequence of SEQ ID NO:10), and having, for example, a histidine buffer with a pH of about 5.8, 6.5% or 8.0% w / v of sucrose, and 0.03% w / v of polysorbate 80.The pharmaceutical composition of the article may contain, in a total volume of about 2 mL to about 25 mL (such as 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), a total amount of about 2500 mg to about 5000 mg (such as 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, such as 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 an antigen-binding fragment thereof (for example, an anti-TTR antibody or an antigen-binding fragment thereof having a VH region and a VL region, the VH region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7 (such as having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 11, and such as having the CDR sequences shown in SEQ ID NOs: 1-3), the VL region containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8 (such as having at least 85%, 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 8, and such as having the CDR sequences shown in SEQ ID NOs: 4-6), and an anti-TTR antibody containing a heavy chain having the amino acid sequence of SEQ ID NO: 9 and a light chain having the amino acid sequence of SEQ ID NO: 10).

[0203] The container can be formed from a variety of materials, such as glass or plastic. The container can be a container that houses the pharmaceutical composition described herein, which composition alone or in combination with another composition is capable of effectively treating or preventing ATTR. The container can have a sterile inlet (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a subcutaneous injection needle). At least one active agent in the composition is the anti-TTR antibody described herein. The label or package insert indicates that the composition is for the treatment of ATTR.

[0204] The article may at least include a first container having a pharmaceutical composition contained therein, wherein the pharmaceutical composition includes the anti-TTR antibody described herein. Optionally, the article may further include a second container having a second therapeutic agent. The article 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 may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as BWFI, PBS, Ringer's solution, and dextrose solution.

[0205] The article may further include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. For example, the article (e.g., a kit) may include a second container having a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and / or glucose or its polymers, such as dextran (e.g., at a concentration of about 5% w / v). The article may include other materials, including buffers, diluents, filters, needles, syringes, and a package insert with instructions for use.

[0206] In a preferred embodiment, the article includes a vial, preferably a clear glass vial sealed with a (gray) rubber stopper and a (blue) aluminum-plastic flip-top cap. Preferably, the antibody is present in the vial at a concentration of 50 mg / mL and is provided as a concentrate for an infusion solution, which appears as a sterile, colorless to light yellow, clear to slightly opalescent liquid, substantially free of visible particles.

[0207] In another preferred embodiment, the article includes a dose syringe or an infusion bag in a metering pump, which contains a diluted antibody preparation.

[0208] Example

[0209] The following examples provided for implementing specific aspects of the present disclosure are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0210] Example 1. Formulation development of anti-TTR monoclonal antibody

[0211] This example summarizes the results of the formulation development of a monoclonal antibody (mAb) against transthyretin (TTR), which contains 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, and is obtained by recombinant expression in CHO-K1 cells respectively. The physical and chemical stability of the anti-TTR antibody was evaluated to design a stable formulation for clinical development. The experiment aimed to evaluate the effect of excipients on the antibody properties under accelerated and stress conditions. Biophysical and analytical methods were used to determine the potential chemical and physical changes that occurred to the antibody formulated under these different conditions.

[0212] The goal of the formulation development study was to identify a stable formulation (e.g., a pharmaceutical composition) of the antibody, as determined according to pH buffer studies, solubility studies, excipient screening studies, and surfactant screening studies.

[0213] Analysis method

[0214] Appearance

[0215] Using a YB-2 light box, the appearance of all samples, including transparency, color, and visible particles, was examined against black and white backgrounds.

[0216] Caliper-SDS R / NR

[0217] Caliper - sodium dodecyl sulfate (SDS) reduction (R) / non - reduction (NR) was performed on a PerkinElmer Caliper automated electrophoresis. The samples were treated with sample buffer, SDS, and N - ethylmaleimide (for non - reduction or NR) or dithiothreitol (for reduction or R) at 70 °C for 10 minutes. Then, through GXII TOUCH TM Samples with a minimum volume of 42 μL (final antibody concentration of 0.045 mg / ml) were tested at excitation / emission wavelengths of 635 nm and 700 nm. The final results were analyzed using commercial software GX Reviewer.

[0218] cIEF

[0219] On a cIEF analyzer equipped with an FC - coated cIEF cassette, capillary isoelectric focusing (cIEF) was performed. Fifty micrograms of each sample was mixed with 90 μl of the master mixture, which consisted of isoelectric point (pI) markers 7.65 / 9.46, PHARMALYTE TM 3 - 10, PHARMALYTE TMIt consists of 8 - 10.5, 1% methylcellulose and 8M urea. After mixing, the samples were focused at 1500V for 1 minute and at 3000V for 8 minutes respectively. The detection wavelength was set at 280nm to evaluate the charge variant distribution within different pI ranges.

[0220] DLS

[0221] The ζ potential was measured by Malvern dynamic light scattering (DLS). The samples were diluted to 5mg / mL, and 1mL of each sample was placed in the DLS sample cell.

[0222] ELISA binding

[0223] The assay was an enzyme - linked immunosorbent assay (ELISA) method for the binding potency of antibodies. Samples and reference standards at appropriate dilutions were loaded onto a 96 - well plate half - area 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 antibodies captured during the previous step. After the final washing step, the TMB substrate solution (Catalog No. N301, THERMO SCIENTIFIC TM ) was loaded into the wells. TMB reacts specifically with peroxide in the presence of peroxidase and generates a colorimetric signal proportional to the amount of antibody bound to the wells. The color development was stopped, and the optical density was measured at 450 / 560nm. Using Pro Software, the dose - response curves of samples, controls, and reference standards were plotted according to a 4 - parameter logistic (auto - estimate) regression model. The EC50 values of each sample and reference standard were calculated separately. The relative binding activity of the samples on the same plate was calculated using the EC50 of the samples relative to the reference.

[0224] Osmolality

[0225] The osmolarity was measured using an osmometer. Before and after testing, the method accuracy of the osmometer was confirmed with a clinitrol 290 milliosmole (mOsm) reference solution. The sample volume used for testing was 20μL, and each sample was tested only once.

[0226] MFI

[0227] Sub - visible particles were monitored by a microfluidic imaging (MFI) system. Approximately 1.5ml volume of each sample was transferred to an MFI 96 - well plate in a biosafety cabinet for analysis. The results were analyzed by the supplier's software. The amount of sub - visible particles in the equivalent circular diameter between over 2μm, over 10μm, and over 25μm was reported.

[0228] Particulates (High Accuracy (HIAC))

[0229] Measure sub-visible particle size and count using a HACH particle analyzer under a laminar flow hood. To avoid introducing air bubbles and interference during inspection, all samples are stored in the hood for at least 0.5 hours before testing. Each sample is tested four times continuously, 0.45 mL each time. The results are expressed as the average number of particles ≥10 μm and ≥25 μm / mL (the method complies with USP <788> Particulate matter in injections).

[0230] pH

[0231] Measure the sample pH using a pH meter with a glass electrode. Calibrate the pH meter before each use.

[0232] Antibody concentration

[0233] Antibody concentration is determined by a THERMO TM UV spectrophotometer. The extinction coefficient used in all evaluation studies is 1.390 AU*mL*mg -1 *cm -1 . All measurements are repeated twice, 2.5 μL of sample each time, and the average result is reported. For concentrations above 100 mg / mL, dilute the sample to 20 mg / mL before testing.

[0234] SEC-HPLC

[0235] Perform size exclusion chromatography (SEC) on a high performance liquid chromatography (HPLC) system with a SEC column (300×7.8 mm, 5 μm). The sampler temperature is set at 5±3 °C, and the column oven temperature is set at 25±3 °C. The mobile phase is 50 mM PB, 300 mM NaCl, pH 6.8±0.1, and the flow rate is set at 1.0 mL / min. Dilute the sample to 10 mg / mL with the mobile phase and inject 100 μg of the sample. The detection wavelength is set at 280 nm, and the run time is 20 minutes. Perform size exclusion chromatography (SEC) on a high performance liquid chromatography (HPLC) system with a SEC column (300×7.8 mm, 5 μm). The sampler temperature is set at 5±3 °C, and the column oven temperature is set at 25±3 °C. The mobile phase is 50 mM PB, 300 mM NaCl, pH 6.8±0.1, and the flow rate is set at 1.0 mL / min. Dilute the sample to 10 mg / mL with the mobile phase and inject 100 μg of the sample. The detection wavelength is set at 280 nm, and the run time is 20 minutes.

[0236] Turbidity (UV350)

[0237] Turbidity is measured by a spectrophotometer (SPECTRA MAX TM ). Add 150 μL of the sample to the wells of a 96-well plate, and also add 150 μL of the corresponding buffer to the corresponding wells as a reference. Then, measure the absorbance of the buffer and the sample at 350 nm. Obtain the UV350 value of the antibody by subtracting the corresponding buffer.

[0238] Viscosity

[0239] Viscosity was measured using a viscometer (Brookfield) and the CP-51 cone and plate geometry was used. Approximately 0.5 mL of sample was used for each measurement and a Brookfield viscosity standard (29.14 cP) was used prior to sample measurement.

[0240] Pre-formulation study

[0241] As detailed below, pH / buffer screening studies and solubility studies were conducted to identify the optimal formulation (e.g., pharmaceutical composition) of the anti-TTR antibody.

[0242] PH / buffer screening study

[0243] The study aimed to screen the pH buffer conditions most suitable for the anti-TTR antibody.

[0244] Sample preparation

[0245] Nine candidate buffers with different pH values were used in this study and the 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 a pH range of 4.5 to 7.5. Drug substance (DS) generated from 50 L of the pool (formulated in 20 mM histidine, pH 6.0) was used for this study. The DS buffer was exchanged to 3 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 using the corresponding acid or base. The anti-TTR antibody concentration used in this study was 50 mg / mL. Each prepared sample was filtered and filled into 2 mL glass vials (1 mL / vial), then stoppered, capped, and immediately labeled.

[0246] Table 2. List of buffer candidates for pH screening

[0247]

[0248]

[0249] Research parameter

[0250] Table 2 shows the evaluation conditions for the pH / buffer screening study. The samples were stored at 40 °C for 4 weeks. Samples were taken out in a timely manner at each time point and kept at 2 °C - 8 °C before analysis. The following test items were performed for this study, including appearance, pH, antibody concentration, SEC-HPLC, cIEF, Caliper-SDS (non-reducing (NR) and reducing (R)), dissociation constant (kD) value and ζ potential by DLS, as further detailed below. For the selected sample F6 at the 4-week (w) time point, ELISA binding antigen was tested.

[0251] Table 3. Study parameters for pH screening study

[0252]

[0253] X = Appearance, pH, antibody concentration (A280), SEC-HPLC, cIEF, Caliper-SDS (NR and R); Y = kD value (DLS), ζ potential; Z = Elisa binding potency (only for F6); w = week

[0254] Results of pH / buffer screening study

[0255] Appearance, pH and protein concentration

[0256] The data summary of appearance, pH and antibody concentration in the pH screening study is listed in Table 4.

[0257] For samples in acetate or histidine buffer (F1 - F6), the samples showed light yellow color, slightly milky white with visible particles at T0, and maintained a similar appearance after incubation at 40 °C for four weeks. Among these six samples, F2 (20 mM acetate pH 5.0) showed a small amount of particles at T0, but no visible particles were shown after incubation at 40 °C later. For F6 (20 mM histidine pH 6.5), a small amount of particles were observed at T0, and a large amount of particles were observed at the 4w point.

[0258] Among these 6 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 turbid in appearance.

[0259] For phosphate buffer samples (F7 - F9), a milky white liquid with a large amount of particles was observed from T0, and the appearance remained unchanged after incubation at 40 °C for four weeks.

[0260] Table 4. Data summary of appearance, pH and antibody concentration in pH screening study

[0261]

[0262] LY = light yellow; SO = light milky white; O = milky white; P = particle; LP = large amount of particles; w = week

[0263] SEC-HPLC

[0264] Table 4 shows the data summary of the SEC-HPLC test results in the pH screening study.

[0265] At time 0, the main peak % range of the samples in acetate or histidine buffer was 97.2% to 97.9%, while the main peak % of the samples in PB buffer was below 96.0%. After four weeks at 40 °C, the histidine buffer (F4-F6) showed the smallest decrease in monomer purity (0.7% - 0.8%) among all the tested buffer systems, while the PB buffer showed the most significant decrease in monomer percentage (up to 3.9%). This is also shown in Figure 1 it.

[0266] After 4 weeks at 40 °C, an increase in high molecular weight (HMW) peaks and an increase in low molecular weight (LMW) peaks were observed in all nine buffers, with the smallest increase in the histidine buffer and the largest increase in the PB buffer.

[0267] Table 5. Data summary of SEC-HPLC in pH screening

[0268]

[0269] ND = not determined; HMW = high molecular weight; LMW = low molecular weight; w = week; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography

[0270] Table 6 shows the data summary of the cIEF test results in the pH / buffer screening study.

[0271] All candidates showed a decrease in the main peak percentage, an increase in acidic peaks and a decrease in basic peaks after storage at 40 °C for 4 weeks. Among all candidates, the histidine buffer had the smallest decrease in the main peak %, ranging from 19.1% to 21.6%, while the PB buffer had the largest decrease in the main peak %, with the cIEF main peak % decreasing by up to 48.1%. The trend of the change in the cIEF main peak % is shown in Figure 2 it.

[0272] Table 6. Data summary of cIEF in pH screening study

[0273]

[0274]

[0275] cIEF = capillary isoelectric focusing; w = week

[0276] Caliper-SDS-(R and NR)

[0277] Table 7 summarizes the data of Caliper-SDS-(R and NR) in the pH buffer screening study. For reducing Caliper-SDS, all candidates showed a decrease in % heavy chain (HC) + light chain (LC)%, and the histidine buffer candidate showed the least decrease in HC + LC% (up to 1.2% decrease) after incubation at 40 °C for 4 weeks. Although the acetate buffer candidate had a slightly greater decrease in HC + LC% (up to 1.7%), however, the HC + LC% of the PB buffer candidate decreased significantly after 4 weeks, with a maximum of 4.7%.

[0278] A similar trend was shown in the non-reducing Caliper-SDS results. After 4 weeks, a decrease in the % of the main peak was shown in all samples, and the histidine buffer showed the least decrease of 2.9% - 3.6%. The main peak % of the acetate buffer decreased by 3.0% to 6.0%, while the PB buffer again showed the most significant change with a decrease in the main peak % of up to 13.1%.

[0279] Table 7. Data summary of Caliper-SDS-(R and NR) in pH buffer screening study

[0280]

[0281]

[0282] SDS = sodium dodecyl sulfate; R = reducing; NR = non-reducing; HC = heavy chain; LC = light chain; w = week

[0283] ELISA binding potency

[0284] After incubation at 40 °C for 4 weeks, the sample (20 mM histidine, pH 6.0) was sent for ELISA binding antigen assay. The results showed that the binding antigen of this sample was 105%, indicating that when formulated in a histidine buffer at pH 6.0, the potency of the anti-TTR antibody did not change significantly after incubation at 40 °C for 4 weeks.

[0285] DLS (kD and ζ potential)

[0286] For all pH buffer candidates, k D values were measured by high-throughput DLS. The ζ potential of all nine samples and the samples added with NaCl was measured by DLS to study whether the addition of NaCl could improve the stability of the antibody. The results are summarized in Table 8.

[0287] Table 8. Summary of data of kD and ζ potential values in the pH buffer screening study

[0288]

[0289]

[0290] k D = dissociation constant; PB = phosphate buffer

[0291] Regarding the kD value, samples in acetate or histidine buffer 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 the PB buffer showed negative values. This indicates that the interaction between antibodies in acetate or histidine buffer is repulsive, thus aggregation is restricted, and antibodies in phosphate buffer showed more attractive interactions, which may lead to more aggregation.

[0292] Regarding the ζ-potential, samples in acetate or histidine buffer showed larger values compared to samples in phosphate buffer, indicating that the antibodies are more stable in these two buffers than in phosphate buffer.

[0293] The ζ-potential value in 20 mM histidine at pH 6.0 was 12.67 mV, and when 200 mM NaCl was added to the buffer, this value decreased to 1.80 mV. This indicates that the addition of NaCl impaired the colloidal stability of the anti-TTR antibody.

[0294] Summary

[0295] Light creamy particles were observed in all pH buffers. At higher pH (e.g., phosphate buffer, pH 6.5, 7.0, and 7.5), a large number of particles and a turbid solution were observed. After incubation at 40 °C for 4 weeks, no significant changes in appearance, pH value, and antibody concentration were observed.

[0296] In acetate or histidine buffer, the kD value and ζ-potential value were higher than those in phosphate buffer, indicating better colloidal stability of the antibody in acetate or histidine buffer. Adding NaCl to the buffer decreased the ζ-potential of the antibody in 20 mM histidine buffer at pH 6.0, which indicates that NaCl did not improve the colloidal stability of the protein.

[0297] Based on the SEC-HPLC results, after storing at 40 °C for 4 weeks, the smallest decrease in the main peak % among all samples was observed in antibodies in histidine buffer (especially at pH 5.5 and 6.0). Antibodies in acetate buffer showed a slightly more decrease in the main peak %, while antibodies in phosphate buffer showed the largest decrease in the SEC-HPLC main peak %.

[0298] The smallest decrease in % of cIEF main peak was detected after 4 weeks of incubation at 40°C for antibodies formulated in histidine buffer (pH 5.5 and 6.0), and the largest decrease was observed for antibodies in phosphate buffer.

[0299] Minimal decreases in reducing Caliper HC+LC% were observed for antibodies in histidine (pH 5.5, 6.0, and 6.5) and acetate buffer (pH 5.0 and 5.5) after incubation at 40°C, while significant decreases in HC+LC% were observed for antibodies in acetate pH 4.5 or phosphate buffer. Regarding non-reducing Calipers: acetate buffer (pH 5.5) and histidine buffer (pH 5.5 and 6.0) showed minimal decreases in IgG purity% after 40°C stress.

[0300] In summary, the histidine pH 5.5 to 6.0 range was identified as providing relatively more stable conditions for the antibody, and the histidine pH 5.8 buffer provided favorable stability.

[0301] Solubility study

[0302] The antibodies were prepared at relatively high concentrations (150 mg / mL) in 20 mM histidine buffer (pH 5.8) with and without NaCl and in phosphate buffered saline (PBS) to study the solubility of the antibodies. Antibody stability was studied at 2°C-8°C and 25°C.

[0303] Sample preparation

[0304] Three preparations were used in the solubility study, the details of which are listed in Table 9. The three preparations were 20 mM histidine pH 5.8, 20 mM histidine pH 5.8 containing NaCl, and PBS. The starting material (e.g., 50 L pool DS) buffer was exchanged into these buffers and concentrated to above 150 mg / mL. All preparation samples were finally filtered with a 0.22 μm polyvinylidene fluoride (PVDF) filter, filled into 2 ml glass vials (1 ml / vial), stoppered and sealed in a biosafety hood.

[0305] Table 9. List of formulation candidates for solubility study

[0306]

[0307] PBS = Phosphate buffered saline

[0308] Research parameter

[0309] Table 10 shows the evaluation conditions for the solubility study. The samples were stored at 25 °C or 5 °C for 2 days (D). Samples were taken at each time point and kept at 2 °C - 8 °C before analysis. The test items included appearance, antibody concentration, turbidity, and viscosity.

[0310] Table 10. Sampling and testing plan for solubility study

[0311]

[0312]

[0313] X = Appearance, protein concentration, turbidity (UV350); Y = Viscosity

[0314] Results of solubility study

[0315] Appearance and antibody concentration

[0316] The appearance, turbidity, and antibody concentration of the solubility study are reported in Table 11. During the study, the samples in 20 mM histidine buffer pH 5.8 (F1) remained light yellow, light milky white, and particle - free. The samples in 20 mM histidine buffer pH 5.8 (F2) containing 155 mM NaCl were light yellow, light milky white, and particle - free at time zero (T0), but became milky white after incubation at 5 °C or 25 °C for 48 hours. The antibody in PBS buffer (F3) was light yellow, milky white, and particle - free during the study. There were a large number of air bubbles in these high - viscosity samples, and it was very difficult to remove these air bubbles.

[0317] During the study, no significant changes were observed in the turbidity or antibody concentration of all samples. The change in turbidity data may be caused by the presence of a large number of air bubbles, and the fluctuation of antibody concentration is caused by dilution changes.

[0318] Table 11. Data summary of appearance, pH, and antibody concentration in the buffer screening study

[0319]

[0320] SY = Light yellow; SO = Light milky white; O = Milky white; FP = Particle - free; H = Hour

[0321] Viscosity

[0322] The sample was diluted to 150 mg / mL for viscosity measurement. The viscosity data are shown in Table 12. All samples showed shear-thinning behavior, and the 20 mM histidine pH 5.8 with NaCl had the lowest viscosity (16 cP), while the 20 mM histidine pH 5.8 (F1) and PBS buffer (F3) had the highest viscosity (about 60 cP).

[0323] Table 12. Viscosity data of solubility study

[0324] Number Formulation Viscosity (cP) F1 20 mM histidine pH 5.8 59.9 F2 20 mM histidine 200 Mm NaCl pH 5.8 16.1 F3 PBS pH 7.4 59.2

[0325] cP = centipoise

[0326] Summary

[0327] The solubility study investigated three different pH buffer types at antibody concentrations above 150 mg / mL. The results showed that no significant changes in antibody concentration or turbidity were observed at 2°C - 8°C and 25°C for 48 hours, indicating the short-term stability of the anti-TTR antibody at a concentration of 150 mg / mL. The 20 mM histidine pH 5.8 buffer showed a favorable appearance among the three studied formulations: light yellow, light milky white, and particle-free.

[0328] At an antibody concentration of 150 mg / mL, the viscosity of the sample was high.

[0329] Based on the above results, the anti-TTR antibody has good stability in 20 mM histidine, pH 5.8 buffer.

[0330] Excipient screening study: Stability at 40 °C and during freeze-thaw

[0331] Sample preparation

[0332] Six formulations were evaluated in the excipient screening, as listed in Table 13. The starting material (e.g., 50 L pool DS) was formulated in 20 mM histidine (pH 5.8) or 20 mM acetate-histidine buffer (pH 5.8). Stock solutions of 40% weight / unit volume (w / v) sucrose, 44.2% (w / v) trehalose 2H2O, 40% (w / v) sorbitol, 40% (w / v) l-arginine hydrochloride, and 10% (w / v) polysorbate 80 (PS80) were prepared. The amounts of DS and excipient stock solutions were calculated and compounded 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 cabinet. An antibody concentration of 100 mg / mL was used in this study.

[0333] Table 13. List of formulation candidates for excipient screening study

[0334]

[0335]

[0336] PS80 = Polysorbate 80

[0337] Research parameter

[0338] Table 14 shows the detailed plan for the excipient screening study. The samples were stored at 40 °C for 4 weeks, or subjected to 3 or 5 freeze - thaw cycles. Samples were taken at each time point and kept at 2 °C - 8 °C before analysis. The test parameters were performed in this study, including appearance, pH, antibody concentration, osmolality, SEC - HPLC, cIEF, Caliper - SDS (reducing and non - reducing), and MFI.

[0339] Table 14. Sampling and Testing Plan for Excipient Screening Study

[0340]

[0341] Test items: X = Appearance, pH, antibody concentration, SEC - HPLC, Caliper - SDS (non - reducing and reducing), cIEF, Y = MFI; Z = Osmolality, kD (DLS); RT = Room temperature

[0342] Results of Excipient Screening 40°C Stability Study

[0343] Appearance, pH, Antibody Concentration, and Osmolality

[0344] The results of appearance, pH, antibody concentration, and osmolality from the 40 °C stability study are presented in Table 15. For the samples without PS 80 in the formulation (F1 - F5), during the 4 - week study, the samples were light yellow and light milky white. The samples at T0 were particle - free, and the samples incubated at 40 °C were particle - free.

[0345] For the samples containing PS80 in the formulation (F6), the samples remained light yellow, light milky white and particle - free during the study, indicating that the addition of PS80 enhanced the stability of the anti - TTR antibody during storage at 40 °C.

[0346] After incubation at 40 °C for 4 weeks, no significant changes in pH value or antibody concentration were observed.

[0347] The osmolality of all samples with excipients ranged from 339 mOsm / kg to 423 mOsm / kg.

[0348] Table 15. Summary of Data on Appearance, pH, Antibody Concentration, and Osmolality in Excipient Screening 40°C Stability Study Summary

[0349]

[0350] SEC-HPLC

[0351] Table 16 shows a data summary of the SEC-HPLC test results in the excipient screening study at 40°C. When stored at 40°C for 4 weeks, except for F4, the percentage of the main peak of all samples decreased by approximately 3.3%, and no significant difference was observed among these five samples. For F4 (with arginine hydrochloride as the excipient), a slightly more significant decrease (3.8%) in the percentage of the main peak was observed after storage at 40°C for 4 weeks.

[0352] Table 16. Summary of SEC-HPLC Data in Excipient Screening Study

[0353]

[0354] HMW = High Molecular Weight; MLW = Low Molecular Weight; w = week; SEC = Size Exclusion Chromatography; HPLC = High Performance Liquid Chromatography

[0355] cIEF

[0356] Table 17 shows a summary of the cIEF test results in the excipient screening study at 40°C.

[0357] When stored at 40°C for 4 weeks, the percentage of the main peak of all samples decreased by approximately 20%, and the percentage of the main peak of F5 showed a slightly greater decrease (25.8%) compared to other formulations. After incubation at 40°C for 4 weeks, a significant increase in the percentage of acidic peaks and a slight decrease in the percentage of basic peaks were also observed.

[0358] Table 17. Summary of cIEF Data in Excipient Screening Study

[0359]

[0360]

[0361] Non-Reducing Caliper-SDS

[0362] Table 18 shows a data summary of the non-reducing Caliper-SDS test results in the excipient screening study at 40°C. At time zero (T0), the purity of all formulations was 97.1%, and it decreased by approximately 3.5% after storage at 40°C for four weeks. No significant difference was observed among the six formulations.

[0363] Table 18. Data summary of non-reducing Caliper-SDS in the excipient screening study

[0364]

[0365] Reducing Caliper-SDS

[0366] Table 19 shows a data summary of the reducing Caliper - SDS results in the excipient screening study at 40°C. At time zero (T0), the purity of all formulations was approximately 99.0%, and it decreased by approximately 1.6% after storage at 40°C for four weeks. No significant differences were observed among the six formulations.

[0367] Table 19. Data summary of reducing Caliper - SDS in excipient screening study

[0368]

[0369]

[0370] HC = heavy chain; LC = light chain; w = week

[0371] Summary of Excipient Screening, 40°C Stability Study

[0372] A large number of bubbles and particles were observed in the samples without PS80. The samples containing 0.02% PS80 were free of visible particles, indicating that PS80 confers stability to the composition by reducing the aggregation of anti - TTR antibodies. All samples were observed to be light yellow and light milky white, and no significant changes were observed in each formulation after incubation at 40°C for 4 weeks.

[0373] After incubation at 40°C for 4 weeks, no significant changes in pH or concentration were observed. The osmolality of all six samples was higher than isotonic.

[0374] For all six formulations, a decrease in SEC purity, cIEF main peak, and Caliper R and NR purity was observed, where F4 showed the largest decrease in SEC purity and F5 showed the highest decrease in the percentage of cIEF main peak.

[0375] No significant differences were observed between F1 and F6, indicating that the addition of PS80 improved the appearance of the samples without reducing the purity of SEC, cIEF, and Caliper R and NR.

[0376] In summary, based on the 40°C stability study, the formulations containing sucrose or sorbitol showed favorable results among all formulations. In addition, the addition of PS80 to the 8% sucrose formulation (F6) did not produce visible particles after incubation at 40°C, indicating that the addition of PS80 enhanced the stability of anti - TTR antibodies during storage at 40°C.

[0377] Results of Excipient Study: Freeze-Thaw Study

[0378] In this excipient study, the stability of all six formulations was evaluated under three and five freeze-thaw cycles. The results are described below.

[0379] Appearance, pH, Antibody Concentration, and Osmolality

[0380] The appearance, pH, and antibody concentration in this freeze-thaw study are presented in Table 20. For samples without PS 80 in the formulation (F1-F5), after the 3 / 5 freeze-thaw cycle study, the samples showed light yellow and light milky white. The samples at T0 were free of particles, and the freeze-thawed samples had particles.

[0381] For the sample containing PS80 in the formulation (F6), the sample remained light yellow, light milky white, and particle-free during the study, indicating that the addition of PS80 enhanced the stability of the anti-TTR antibody during the freeze-thaw cycle.

[0382] After up to 5 freeze-thaw cycles, no significant changes in the pH value or antibody concentration were observed.

[0383] Table 20. Appearance, pH, and Antibody Concentration in Freeze-Thaw Study of Excipient Screening

[0384]

[0385] SY: Light yellow, SO: Light milky white, P: Particle, FP: Particle-free

[0386] SEC-HPLC

[0387] Table 21 shows a summary of the SEC-HPLC test results in the excipient screening freeze-thaw study. After ≤ 5 freeze-thaw cycles, no significant changes were observed in SEC-HPLC Main. - For F4 (arginine hydrochloride as excipient), a slight decrease (0.9%) in the main peak percentage was observed after 5 freeze-thaw cycles.

[0388] Table 21. Summary of SEC-HPLC Data in Excipient Screening Study

[0389]

[0390]

[0391] cIEF

[0392] Table 22 shows a summary of the cIEF test results in the excipient screening study.

[0393] After 5 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 (acetic acid-histidine buffer, sucrose as excipient), respectively. After up to five freeze-thaw cycles, no significant changes were observed in other formulations.

[0394] Table 22. Summary of cIEF5 Data in Excipient Screening Study

[0395]

[0396] Non-Reducing Caliper-SDS

[0397] Table 23 shows a 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 significant change in purity was observed after 5 freeze - thaw cycles.

[0398] Table 23. Data summary of non - reducing Caliper - SDS in excipient screening study

[0399]

[0400]

[0401] Reducing Caliper-SDS

[0402] Table 24 shows a data summary of the reducing Caliper - SDS test results in the excipient screening study. At time zero (T0), the purity of all formulations was approximately 99.0%, and there was no significant decrease after 5 freeze - thaw cycles.

[0403] Table 24. Data summary of reducing Caliper - SDS in excipient screening freeze - thaw study

[0404]

[0405] HC = heavy chain; LC = light chain

[0406] Subvisible Particles (MFI)

[0407] Sub - visible particles measured by MFI in the excipient screening study are shown in Table 25. For the formulations without PS80 (F1 - F5), a large number of sub - visible particles were detected starting from time zero (T0), while for the sample containing PS80 (F6), the number was much lower. The addition of PS80 significantly reduced the number of sub - visible particles in the sample. No significant change was observed in all formulations before and after 5 freeze - thaw cycles.

[0408] Table 25. Subvisible Particles Measured by MFI in Freeze-Thaw Study of Excipient Screening

[0409]

[0410]

[0411] MFI = Microfluidic Imaging; ECD = Equivalent Circular Diameter; AR = Aspect Ratio

[0412] Summary of Freeze-Thaw Study

[0413] In the sample without PS80, it is difficult to distinguish a large number of bubbles and particles, while the sample containing 0.02% PS80 contains no visible particles, indicating that PS80 imparts stability to the composition by reducing the aggregation of anti-TTR antibodies. All samples were observed to be light yellow and light milky white, and after 5 freeze-thaw cycles, no significant changes were observed in each formulation. A large number of subvisible particles were detected in the formulation without PS80, while a significant reduction in subvisible particles was observed when PS80 was added to the formulation.

[0414] After 5 freeze-thaw cycles, no significant changes in pH or concentration were observed.

[0415] For F4 (arginine hydrochloride as excipient), a 0.9% decrease in the percentage of the SEC main peak was observed, while for the other five formulations, significant changes in the percentage of the SEC main peak were observed. Regarding cIEF, F3 (sorbitol as excipient) or F5 (acetic acid-histidine as buffer) showed a slightly decreased percentage of the main peak, while for the other formulations, no significant changes in the cIEF main peak % were observed after 5 freeze-thaw cycles. Comparing all excipients, the formulation containing sucrose had the best antibody stability in 20 mM histidine buffer.

[0416] After 5 freeze-thaw cycles, no significant changes in Caliper NR and R were observed for all samples.

[0417] No significant differences were observed between F1 and F6, indicating that the addition of PS80 improved the appearance and subvisible particles of the sample without reducing the purity of SEC, cIEF, and Caliper R and NR.

[0418] In summary, the formulation containing sucrose and PS80 performed the best among all formulations. For example, the addition of PS80 to an 8% sucrose formulation (F6) did not produce visible particles after one or more freeze-thaw cycles, indicating that the addition of PS80 enhanced the stability of anti-TTR antibodies during storage at 40 °C.

[0419] Summary of Excipient Screening Study

[0420] To summarize the 40 °C stability and freeze-thaw studies, it was observed that the formulation containing sucrose was the most stable for the antibody, indicated by the lowest reduction in SEC, cIEF, and Caliper purity compared to other formulations. The addition of PS80 did improve the appearance and reduce subvisible particles, with essentially no negative impact on antibody stability.

[0421] A 20 mM histidine buffer, 8% or 6.5% (w / v) sucrose, and a pH of 5.8 were selected as the final buffer and excipient system. The surfactant (PS80) concentration will be evaluated in the next study.

[0422] Surfactant Screening Study

[0423] The purpose of this study was 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).

[0424] Sample Preparation

[0425] Six formulations were evaluated in the surfactant screening as listed in Table 26. The starting material (e.g., 50 L pool DS) was buffer-exchanged into 20 mM histidine (pH 5.8). A stock solution of 60% (w / v) sucrose and 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 recipe. 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 cabinet. Note that all formulations except the freeze-thaw samples were used with 2R vials, and the freeze-thaw samples were filled into 6R samples because the 2 mL in the 2R vials caused vial breakage during freeze-thaw.

[0426] Table 26. List of Formulation Candidates for Surfactant Screening Study

[0427]

[0428] PS80 = polysorbate 80

[0429] Study Parameters

[0430] Table 27 shows the sampling and testing plan for the surfactant screening study. The samples were agitated at 200 rpm at 25 °C for 3 or 7 days (D), or were also evaluated without agitation at 25 °C for 3 or 7 days. For the freeze-thaw study, for each cycle, the samples were frozen completely at -70 °C and thawed completely at room temperature, and 3 or 5 cycles were evaluated. The samples were also held at 40 °C for up to 2 weeks. Samples were taken in a timely manner and held at 2 °C - 8 °C prior to analysis. For this study, test items were performed, including appearance, pH, antibody concentration, osmolarity, SEC-HPLC, Caliper-SDS (reducing and non-reducing), cIEF, and subvisible particles (HIAC).

[0431] Table 27. Sampling and Testing Plan for Surfactant Screening Study

[0432]

[0433] X = Appearance, pH, antibody concentration, SEC-HPLC, non-reducing and reducing Caliper-SDS, cIEF, HIAC; Y = Osmolality; D = Day

[0434] Results

[0435] Appearance, pH, Osmolality, and Antibody Concentration

[0436] Table 28 shows the data summary of appearance, pH, osmolality and antibody concentration of this study. After stirring at 200 rpm for 7 days, all samples remained light yellow, light milky white and free of particles.

[0437] The pH and antibody concentration of F1 - F6 remained stable during the study, with the pH value being 5.8 - 5.9 and the antibody concentration being approximately 100 mg / mL.

[0438] The osmolality of F1 - F3 (8% sucrose) was approximately 340, while that of F4 - F6 (6.5% sucrose) was approximately 280. Therefore, F1 - F3 and F4 - F6 meet the European Pharmacopoeia limit of > 240 mOsm / kg.

[0439] Table 28. Summary of Data on Appearance, pH, Osmolality, and Antibody Concentration in Surfactant Screening Stirring Study Summary

[0440]

[0441]

[0442] SY: Light yellow, SO: Light milky white; D = Day

[0443] SEC-HPLC

[0444] The SEC-HPLC results of the stirring study in surfactant screening are reported in Table 29. After stirring for 7 days, the percentage of the main peak of all test samples remained stable, and no significant differences were observed among these six samples.

[0445] Table 29. Summary of SEC-HPLC Data in Surfactant Screening Stirring Study

[0446]

[0447] D = Day; HMW = High molecular weight; LMW = Low molecular weight

[0448] cIEF

[0449] The cIEF results of the surfactant screening stirring study are reported in Table 30. After stirring at 200 rpm for 7 days, no significant change in the percentage of the cIEF main peak was observed in all six formulations.

[0450] Table 30. Summary of cIEF Data in Surfactant Screening Stirring Study

[0451]

[0452] Non-Reducing Caliper-SDS

[0453] The non-reducing Caliper-SDS results are reported in Table 31. The non-reducing Caliper-SDS purity percentages of all test samples remained stable, and no significant differences were observed among these samples during the study.

[0454] Table 31. Summary of Non-Reducing Caliper-SDS Data in Surfactant Screening Stirring Study

[0455]

[0456] D = days

[0457] Reducing Caliper-SDS

[0458] The reducing Caliper-SDS results of the surfactant screening stirring study are reported in Table 32. The reducing Caliper SDS purity percentages of all test samples remained stable, and no significant differences were observed among these samples during the study.

[0459] Table 32. Summary of Reducing Caliper-SDS Data in Surfactant Screening Stirring Study

[0460]

[0461] HC = heavy chain; LC = light chain; D = days

[0462] Subvisible Particles (HIAC)

[0463] Table 33 shows a data summary of the subvisible particle (HIAC) test results of the stirring study in surfactant screening. After stirring for 7 days, no significant change in subvisible particles was observed in the six formulations.

[0464] Table 33. Summary of Subvisible Particles (HIAC) Data in Stirring Study

[0465]

[0466] D = days

[0467] Summary of Stirring Study

[0468] Up to 7 days of agitation was used in the surfactant screening study to evaluate the stability of the formulations. No significant differences were observed among the six different formulations after the stress test.

[0469] 25°C Stability

[0470] Appearance, pH, and Antibody Concentration

[0471] Table 34 shows the data summary of the appearance, pH, and antibody concentration of this study. All samples were light yellow, light milky white, and free of particles, and no significant changes were observed after incubation at 25 °C for 7 days.

[0472] The pH and antibody concentration of F1 - F6 remained stable during the study, with the pH value being 5.8 - 5.9 and the antibody concentration being approximately 100 mg / mL.

[0473] Table 34. Summary of Data on Appearance, pH, and Antibody in Surfactant Screening 25°C Study

[0474]

[0475]

[0476] D = day

[0477] SEC-HPLC

[0478] The SEC - HPLC results of the surfactant screening study at 25 °C are reported in Table 35. After incubation at 25 °C for 7 days, the percentage of the main peak of all tested samples remained stable, and no significant differences were observed among these samples.

[0479] Table 35. Summary of SEC-HPLC Data in Surfactant Screening 25°C Study

[0480]

[0481] HMW = high molecular weight; LMW = low molecular weight; w = week; SEC = size - exclusion chromatography; HPLC = high - performance liquid chromatography; D = day

[0482] cIEF

[0483] The cIEF results of the surfactant screening study at 25 °C are reported in Table 36. After incubation at 25 °C for 7 days, no significant changes were observed in the percentage of the cIEF main peak of all six formulations.

[0484] Table 36. Summary of cIEF Data in Surfactant Screening 25°C Study

[0485]

[0486] cIEF = capillary isoelectric focusing; D = day

[0487] Non-Reducing Caliper-SDS

[0488] The non-reducing Caliper-SDS results of the surfactant screening study at 25 °C are reported in Table 37. The non-reducing Caliper-SDS purity percentages of all test samples remained stable, and no significant differences were observed among these samples during the study.

[0489] Table 37. Summary of Non-Reducing Caliper-SDS Data in Surfactant Screening Study

[0490]

[0491] SDS = sodium dodecyl sulfate; D = day

[0492] Reducing Caliper-SDS

[0493] The reducing Caliper-SDS results are reported in Table 38. The reducing Caliper SDS purity percentages of all test samples remained stable, and no significant differences were observed among these samples during the study.

[0494] Table 38. Summary of data for reducing Caliper-SDS in the surfactant screening study

[0495]

[0496] SDS = sodium dodecyl sulfate; D = day; HC = heavy chain; LC = light chain

[0497] Subvisible Particles (HIAC)

[0498] Table 39 shows a summary of the data for the subvisible particle (HIAC) tests of the 25 °C study in the surfactant screening. After incubation at 25 °C for 7 days, no significant changes in subvisible particles were observed in the six formulations.

[0499] Table 39. Summary of Subvisible Particles (HIAC) Data in 25°C Study

[0500]

[0501] HIAC = High Accuracy; D = Day

[0502] Summary of 25°C Study

[0503] The results showed that no significant changes were observed in appearance, pH, antibody concentration, SEC-HPLC main peak %, cIEF main peak %, or subvisible particles (HIAC) for all six formulations after incubation at 25 °C for up to 7 days.

[0504] Freeze-Thaw Study

[0505] In surfactant screening studies, up to 5 cycles of freeze - thaw studies were used to evaluate the stability of the formulation.

[0506] Appearance, pH, and Antibody Concentration

[0507] Table 40 shows a summary of the data on the appearance, pH, and antibody concentration of the freeze - thaw studies. After 5 freeze - thaw cycles, all samples showed a light yellow, light milky white appearance and were free of particles.

[0508] The pH and antibody concentration of F1 - F6 remained stable during the study, with a pH value of 5.8 - 5.9 and an antibody concentration of approximately 100 mg / mL.

[0509] Table 40. Summary of data on appearance, pH, and antibody in surfactant screening freeze - thaw studies

[0510]

[0511]

[0512] SY = light yellow; SO = light milky white; O = milky white; FP = free of particles; D = day

[0513] SEC-HPLC

[0514] The SEC - HPLC results are reported in Table 41. After 5 freeze - thaw cycles, the percentage of the main peak of all test samples remained stable, and no significant differences were observed among these samples.

[0515] Summary of SEC-HPLC data in surfactant screening freeze-thaw study

[0516]

[0517] HMW = high molecular weight; MLW = low molecular weight; w = week; SEC = size - exclusion chromatography; HPLC = high - performance liquid chromatography; D = day

[0518] cIEF

[0519] The cIEF results are reported in Table 42. After 5 freeze - thaw cycles, no significant changes in the percentage of the cIEF main peak were observed in all six formulations.

[0520] Summary of cIEF data in surfactant screening freeze-thaw study

[0521]

[0522] cIEF = capillary isoelectric focusing; C = cycle

[0523] Non-reducing Caliper-SDS

[0524] The non-reducing Caliper-SDS results are reported in Table 43. The non-reducing Caliper-SDS purity percentages for all test samples remained stable and no significant differences were observed among these samples during the study.

[0525] Summary of Non-reducing Caliper-SDS data in surfactant screening freeze-thaw study

[0526]

[0527] SDS = sodium dodecyl sulfate

[0528] Reducing Caliper-SDS

[0529] The reducing Caliper-SDS results are reported in Table 44. The reducing Caliper SDS purity percentages for all test samples remained stable and no significant differences were observed among these samples during the study.

[0530] Summary of Reducing Caliper-SDS data in surfactant screening freeze-thaw study

[0531]

[0532] SDS = sodium dodecyl sulfate; HC = heavy chain; LC = light chain

[0533] Subvisible particles (HIAC)

[0534] Table 45 shows a data summary of the subvisible particle (HIAC) test results in the surfactant screening freeze-thaw study. After 5 freeze-thaw cycles, no significant changes in subvisible particles were observed in the six formulations.

[0535] Summary of Subvisible particles (HIAC) data in surfactant screening freeze-thaw study

[0536]

[0537] * Due to vial breakage during freeze-thaw, the sample volume was insufficient for HIAC testing.

[0538] Summary of freeze-thaw study

[0539] The results showed that after up to 5 freeze-thaw cycles, no significant changes were observed in appearance, pH, antibody concentration, SEC-HPLC main peak %, cIEF main peak %, or subvisible particles (HIAC) for all six formulations.

[0540] 40°C study

[0541] Surfactant screening studies were performed using a two-week incubation at 40 °C to evaluate the stability of these six formulations.

[0542] Appearance, pH and antibody concentration

[0543] Table 46 shows a summary of the data for appearance, pH, and antibody concentration for the 40 °C study. After incubation at 40 °C for 2 weeks, all samples remained light yellow, light creamy white, and particle-free.

[0544] The pH and antibody concentration of F1 - F6 remained stable during the study, with a pH value of 5.8 - 5.9 and an antibody concentration of approximately 100 mg / mL.

[0545] Summary of Appearance, pH and antibody data in surfactant screening 40°C study

[0546]

[0547] SY = light yellow; SO = light creamy white; O = creamy white; FP = particle-free; w = week

[0548] SEC-HPLC

[0549] The SEC-HPLC report of the surfactant screening 40 °C study is shown in Table 47. After incubation at 40 °C for 2 weeks, a decrease in the percentage of the main peak was observed in all six samples, as Figure 3 shown. Figure 4 shows different decreases in the main peak % in these six formulations, and a more significant decrease in the SEC main peak % was observed with an increase in the PS80 concentration.

[0550] Summary of SEC-HPLC data in surfactant screening 40°C study

[0551]

[0552] HMW = high molecular weight; MLW = low molecular weight; w = week; SEC = size exclusion chromatography; HPLC = high performance liquid chromatography

[0553] cIEF

[0554] The cIEF results of the surfactant screening 40 °C study are reported in Table 48. After incubation at 40 °C for 2 weeks, a significant decrease in the main peak % (about 12%) was observed in all formulations, and no obvious differences were observed among the six formulations.

[0555] Summary of cIEF data in surfactant screening 40°C study

[0556]

[0557] cIEF = capillary isoelectric focusing; w = week

[0558] Non-reducing Caliper-SDS

[0559] The results of non-reducing Caliper-SDS for surfactant screening studies at 40 °C are reported in Table 49. The non-reducing CE-SDS purity decreased by approximately 1.8%, and no significant differences were observed between these samples during the study.

[0560] Summary of Non-reducing Caliper-SDS data in surfactant screening 40°C study

[0561]

[0562] SDS = sodium dodecyl sulfate; w = week

[0563] Reducing Caliper-SDS

[0564] The results of reducing Caliper-SDS are reported in Table 50. No significant changes in reducing Caliper SDS were observed in these samples during the study.

[0565] Summary of Reducing Caliper-SDS data in surfactant screening 40°C study

[0566]

[0567] SDS = sodium dodecyl sulfate; HC = heavy chain; LC = light chain

[0568] Subvisible particles (HIAC)

[0569] Table 51 shows a data summary of the subvisible particle (HIAC) test results for the 40 °C studies in surfactant screening. No significant changes in subvisible particles were observed in the six formulations after incubation at 40 °C for 2 weeks.

[0570] Summary of Subvisible particles (HIAC) data in 25°C study

[0571]

[0572] Summary of 40°C study

[0573] The results showed that no significant changes were observed in appearance, pH, antibody concentration, reducing Caliper, or subvisible particles (HIAC) for all six formulations after incubation at 40 °C for up to 2 weeks. A significant decrease in the SEC-HPLC main peak % and cIEF main peak % was observed after incubation at 40 °C. An increase in the PS80 concentration from 0.02% to 0.06% resulted in a decrease in the SEC main peak % after incubation at 40 °C for 2 weeks.

[0574] Summary

[0575] No significant 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.

[0576] In the 40°C stress study, a decrease in SEC main peak % and cIEF main peak % was observed. The SEC purity of the samples containing PS80 decreased with the increase of PS80, so a lower concentration of PS80 is recommended. Since the range of 0.02% and 0.04% PS80 was studied, a concentration of 0.03% PS80 is recommended for the formulation.

[0577] In summary, the target formulation of the anti-TTR antibody is 20 mM histidine buffer at pH 5.8, 8% (w / v) sucrose, and 0.03% (w / v) PS80.

[0578] Example 2. Stability study of anti-TTR monoclonal antibody formulations

[0579] As outlined in detail in Example 1 above, the formulation development of NI006 / ALXN2220 includes studies designed to select buffer systems and excipients to stabilize the protein. Formulations were developed to prevent product loss and minimize the decline in purity and bioactivity in resistance to stresses encountered during production, storage, transportation, and handling.

[0580] pH buffer screening studies were conducted to determine the optimal buffer system for the drug product formulation. 20 mM histidine buffer at pH 5.8 was selected as the final buffer system.

[0581] Different types of excipients were evaluated through excipient studies, including disaccharides (such as sucrose and trehalose), amino acids (such as L-arginine hydrochloride), polyols (such as sorbitol), and surfactants (such as polysorbate 80). The samples were incubated at 40°C for up to 4 weeks. Thermal stability, the 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 the decrease in SEC, cIEF, and caliper purity, thus maintaining product purity.

[0582] In the excipient concentration screening study, three different concentrations of polysorbate 80 (0.02%, 0.04%, and 0.06% (w / v)) and two concentrations of sucrose (6.5% and 8% (w / v)) were ultimately tested. Three stress conditions were used in the screening: agitation, heat, and freeze-thaw. In the agitation study, the samples were placed at 25°C and agitated at 200 rpm or not agitated for up to 7 days. In the heat study, the samples were placed at 40°C for up to 2 weeks. In the freeze-thaw study, the samples were freeze-thawed up to 5 cycles. Appearance, pH, protein concentration, number of subvisible particles, and purity were evaluated.

[0583] A concentration of 0.03% (w / v) of polysorbate 80 was selected as the surfactant strength because subvisible particle formation was effectively inhibited and high SEC purity was maintained. A comparative study was conducted to compare two formulations with sucrose concentrations of 6.5% or 8% (w / v). After incubation at 40°C for 1 month or at 25°C for 3 months, no significant differences were observed between the formulations of 6.5% and 8% (w / v) sucrose in terms of DSC (differential scanning calorimetry), appearance, pH, protein concentration, SEC, cIEF, CE-SDS (non-reducing and reducing), subvisible particles, and potency.

[0584] The final formulated product developed was NI006 / ALXN2220 at a target concentration of 50 mg / mL in 20 mM histidine buffer, 8% (w / v) sucrose, 0.03% (w / v) polysorbate 80, and a pH of 5.8. Excipients were selected based on their stabilizing effects on the drug product. L-histidine and L-histidine monohydrochloride at a concentration of 20 mM stabilized the pH in the liquid state. Sucrose at a concentration of 8% (w / v) changed the osmolarity to isotonic, stabilized the NI006 / ALXN2220 protein to prevent aggregate formation in the liquid state, and acted as a cryoprotectant during freezing / thawing. Polysorbate 80 at a concentration of 0.03% (w / v) was selected to stabilize the NI006 / ALXN2220 protein and prevent surface-induced protein denaturation or aggregation in the liquid state.

[0585] Manufacturing process development

[0586] The pharmaceutical manufacturing process consists of thawing of drug substance, combining and mixing, aseptic filtration, aseptic filling, stoppering, capping, visual inspection, and batch packaging. Aseptic filtration is selected as the method to obtain a sterile drug product and is carried out by using two aseptic filters (0.22 μm, PVDF) connected in series. Before and after aseptic filtration, the filters are subjected to a bubble point test with water to ensure filter integrity. The compatibility of the drug product with the contact components on the filling line, the effect of shear stress caused by peristaltic pumps, and the stability under light exposure have been evaluated to mitigate potential adverse effects on product quality attributes during manufacturing. Non-clinical batches (batch 201901004) and three clinical batches (batch 201903038, batch 201904050, and 20200801) have been filled. The non-clinical batches and clinical batches use the same filling volume, container closure system, unit operation sequence, and storage conditions. There are no significant changes in the pharmaceutical manufacturing process between the non-clinical batches and the clinical batches. Minor differences are described below:

[0587] · A 2 L scale is used for non-clinical batches and a 14 L scale is used for clinical batches.

[0588] · The formulation for non-clinical batch 201901004 is 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8). The formulations for clinical batches 201903038 and 201904050 are 50 mg / mL in 20 mM histidine buffer, 6.5% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8). The formulation for clinical batch 20200801 is 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) (pH 5.8).

[0589] · 5 L glass bottles are used for combining and mixing of drug substance for non-clinical batches and 50 L mixing bags are used for combining and mixing of drug substance for clinical batches.

[0590] Thawing, pooling and mixing of bulk drug substance

[0591] The frozen drug substance stored in 2 L PETG bottles is thawed at room temperature (18 °C - 24 °C) in a room protected from light. After complete thawing, the drug substance is combined into a 50 L mixing bag and stirred at an appropriate speed so that movement is observed without generating foam. The mixing time is controlled between 15 minutes and 20 minutes. Before aseptic filtration, samples are taken for pH, protein concentration, osmolality, and bioburden testing.

[0592] Sterile filtration

[0593] In a Class A environment, the bulk drug substance is aseptically filtered through two serially connected 0.22 μm sterile filters via a peristaltic pump into a sterile 20 L disposable bag. Filter integrity tests are performed on the two filters before and after aseptic filtration.

[0594] Sterile filling

[0595] Aseptic filling is carried out inside a Restricted Access Barrier System (RABS) unit which completely encloses the filler and provides a Class A environment. The RABS unit separates the operator from the aseptic interior. All filling components are autoclaved and aseptically assembled. Sterile, pyrogen-free 2 mL (2R) glass vials are filled to a target volume of 2.25 mL. Filling weight checks are performed periodically during filling to ensure that the filling weight is 2.233 - 2.442 g / vial.

[0596] Stoppering

[0597] Inside the RABS unit, the filled vials are automatically stoppered with 13 mm rubber stoppers. The stoppers are steam sterilized at 122 °C for 30 minutes.

[0598] Capping

[0599] Under Class A laminar protection, the stoppered vials are transferred via a conveyor belt to a capping machine. The stoppered vials are capped with 13 mm plastic-aluminum flip-off caps. The caps are steam sterilized at 122 °C for 30 minutes.

[0600] Visual inspection

[0601] The capped vials are manually 100% visually inspected by production personnel and then subjected to a statistically based Acceptable Quality Limit (AQL) inspection by Quality Assurance. Release and stability samples are taken after visual inspection.

[0602] Bulk packaging and storage

[0603] Then the filled drug product vials are bulk packaged and labeled. The bulk packaged drug product vials are stored at 2 °C - 8 °C.

[0604] Container closure system

[0605] The container closure system for the drug product is a 2 mL (2R) Type I glass vial sealed with a 13 mm rubber stopper and a 13 mm aluminum flip-off cap. Components are selected that are durable to the sterilization and depyrogenation processes, and non-reactive contact surfaces that are optimally compatible with the protein. The compatibility of the container closure system with the drug product is evaluated by the accelerated and long-term stability studies given below.

[0606] The integrity of the container closure system is demonstrated by dye ingress testing. The drug product vials are immersed under a colored dye and held under vacuum, then the vacuum is released and the penetration of the dye into the vials is examined. The container and closure system achieve 100% airtightness. Container closure integrity testing (CCIT) is performed annually in the stability program using a non-destructive vacuum decay method. The product contact materials, glass vials, and rubber stoppers have been tested according to USP and European Pharmacopoeia requirements and are suitable for parenteral use. Compliance with each batch of vials and stoppers is verified on the certificate of compliance provided by the supplier.

[0607] Analytical procedures

[0608] Color

[0609] The color complies with European Pharmacopoeia 2.2.2. Color measurement is performed using a color difference method.

[0610] Transparency

[0611] The transparency complies with European Pharmacopoeia 2.2.1. Transparency measurement is performed using a light scattering method.

[0612] pH

[0613] Complies with USP <791> and European Pharmacopoeia 2.2.3. pH measurement is performed using a potentiometric method.

[0614] Osmolality

[0615] Complies with USP <785> and European Pharmacopoeia 2.2.35. Osmolality is indirectly determined by measuring the depression of the freezing point of the solution.

[0616] iCIEF

[0617] Integrated Capillary Isoelectric Focusing (iCIEF) is an identity and purity analysis method used to separate proteins based on their isoelectric point (pI) and monitor the percentage of charge variant species in a protein sample. The pI is an inherent property of a specific protein molecule and is the pH at which the protein molecule carries no net charge. Under an external electric field, charge variants move along a continuous pH gradient formed by ampholytes and stop when the pH equals their pI. At this pI, the protein carries no net charge and is not attracted to either electrode. Thus, different monoclonal antibody species with different pI values are separated and focused at different positions. The pI values and relative abundances of resolvable peaks can be identified and quantified using chromatography software.

[0618] To meet the acceptance criteria of "characteristics consistent with the reference standard", the electrophoretogram should show peak shapes comparable to those of the reference standard. In addition, the difference (average value) in the pI values of the main peaks between the test sample and the reference standard should not exceed 0.2.

[0619] SEC-HPLC

[0620] Size exclusion chromatography - high performance liquid chromatography (SEC-HPLC) is a purity analysis method for separating proteins based on the size of the proteins. The stationary phase consists of inert particles packed into a dense three-dimensional matrix. The particles have small pores that only allow substances below a certain size to enter. Larger molecules simply pass over the pores because they are too large to enter. Thus, larger molecules flow through the column faster; the smaller the molecule, the longer the retention time. After separation, the relative percentages of high molecular weight (HMW) substances, monomers, and low molecular weight (LMW) substances are quantified via UV detection.

[0621] CE-SDS (reducing)

[0622] Reducing capillary electrophoresis - sodium dodecyl sulfate (CE-SDS) is a purity analysis method for separating proteins based on the electrophoretic mobility of the proteins, where smaller-sized proteins move faster than larger-sized 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 β-mercaptoethanol (BME) to the sample solution. Separation is carried out through an uncoated capillary, and the protein sample is detected at 220 nm using a photodiode array (PDA) detector. The results are reported as a percentage of purity.

[0623] CE-SDS (non-reducing)

[0624] CE-SDS (non-reducing) is a purity analysis method for separating proteins based on the electrophoretic mobility of the proteins, where smaller-sized proteins move faster than larger-sized proteins. In this method, the test sample is denatured by heating in the presence of SDS. The alkylating agent N-ethylmaleimide (NEM) is added to the sample solution to prevent sulfhydryl groups from binding to other sulfhydryl groups. Separation is carried out through an uncoated capillary, and the protein sample is detected at 220 nm using a PDA detector. The results are reported as a percentage of purity.

[0625] Bioburden

[0626] The bioburden test is performed by membrane filtration based on USP <61> and European Pharmacopoeia 2.6.12. A 10 mL sample of the drug substance is surface-filtered aseptically through a 0.45 μm membrane. The filter membrane is then transferred to a culture plate of soybean-casein digest agar for determination of the total aerobic microbial count (TAMC). Another filter membrane used for filtration of the 10 mL drug substance sample is transferred to a culture plate of Sabouraud dextrose agar for determination of the total combined yeast and mold count (TYMC).

[0627] Endotoxin

[0628] The bacterial endotoxin test is performed by kinetic turbidimetry and is established based on USP <85> and European Pharmacopoeia 2.6.14. An amoebocyte lysate from horseshoe crabs is used to detect endotoxins produced by Gram-negative bacteria, which coagulates together with the endotoxins. By establishing the correlation between the endotoxin concentration and the time required to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development, the endotoxin concentration can be calculated.

[0629] ELISA (binding assay)

[0630] The binding potency of the NI006 / ALXN2220 antibody is evaluated using the ELISA method. Samples, controls, and reference standards at appropriate dilutions are loaded onto a 96-well plate with half areas coated with misfolded TTR (the antigen of NI006 / ALXN2220). After washing the plate, horseradish peroxidase (HRP)-conjugated goat anti-human IgG is added to the wells to allow interaction with the bound NI006 / ALXN2220 antibody captured by the misfolded TTR. After the final washing step, a TMB substrate solution is loaded into the wells. TMB reacts specifically with peroxide in the presence of peroxidase and generates a colorimetric signal proportional to the amount of NI006 / ALXN2220 protein bound to the wells. The color development is stopped, and the optical density is measured at 450 nm (subtracting 560 nm for wavelength correction).

[0631] Using SoftMax Pro GxP software, sample and reference standard dose-response curves are plotted according to a 4-parameter logistic (auto-estimation) regression model. The respective EC50 values of the samples and reference standards are calculated. The relative binding activity of the sample is calculated using the following formula:

[0632] Relative binding activity of the sample (%) =

[0633] (EC50 of reference standard / EC50 of sample) × 100%

[0634] Protein concentration

[0635] Because aromatic amino acids are present in protein molecules, proteins in solution absorb ultraviolet light with a wavelength of 280 nm. 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 extinction coefficient (ε) of the protein as follows: A = Clε. Different from traditional UV-Vis methods that rely on a single absolute absorbance value, slope spectrometry uses cross-sectional data (absorbance versus path length) to determine a slope value in order to quantify the sample concentration using a slope spectrometry equation (Slope = εC) derived from the Beer-Lambert law.

[0636] Cell-based assay

[0637] 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 2×104 THP-1 cells / well in assay medium were seeded at 100 μL / well into 96-well cell culture plates, and then mixtures of serial dilutions of NI006 / ALXN2220 mAb standards (final concentrations: 2000 - 0.039 g / mL) and misfolded TTR (final concentration: 10 μg / mL) were loaded into the wells in duplicate at 100 μL / well. After incubation at 37 °C and 5% CO2 for 20 - 24 hours, IL-8 production was measured using a human IL-8 ELISA kit.

[0638] Compatibility study

[0639] Evaluate the compatibility of NI006 / ALXN2220 with materials in clinical use. Assess the compatibility with the following clinical administration settings:

[0640] · Bags and infusion sets under PVC settings - IV bags, IV lines, and filters are made of PVC materials

[0641] · Bags and infusion sets under non-PVC settings - IV bags, IV lines, and filters are made of non-PVC materials

[0642] · Syringes under PVC settings - Infusion lines and filters are made of PVC materials

[0643] · Syringes under non-PVC settings - Infusion lines and filters are made of non-PVC materials

[0644] Test at three concentrations (1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL) for 24 hours at 2°C - 8°C and then for 6 hours at 25°C (30 hours in total). Use saline and glucose as diluents for concentrations of 1.0 mg / mL and 20.0 mg / mL. The results are shown in Tables 52 and 53.

[0645] Table 52. Results of in-use compatibility study 。

[0646]

[0647]

[0648] Cl: Colorless; SY: Light yellow; C: Clear; FoP: Free of visible particles; SO: Light milky white

[0649] 1 The concentration is the same as that of the NI006 / ALXN2220 drug product.

[0650] Table 53. Results of in-use compatibility study (continued)

[0651]

[0652]

[0653]

[0654] 1 The concentration is the same as that of the NI006 / ALXN2220 drug product.

[0655] 2 The variability of the results is due to the variability of the assay. All results of the in-use compatibility study are within the acceptance criteria. Considering that within the short in-use time frame, the most likely incompatibilities with plastic materials would be adsorption (addressed by protein concentration) and visible particle formation (addressed by pharmacopeial methods), the results are not considered safety-related.

[0656] In the saline group, visible particles were observed, indicating that NI006 / ALXN2220 is less stable in saline. The data show that when using glucose as a diluent, no significant changes were observed in appearance, protein concentration, sub-visible particles, SEC-HPLC, and ELISA binding assays. 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°C - 8°C and then for 6 hours at 25°C (30 hours in total). NI006 / ALXN2220 is compatible with the materials evaluated for clinical use.

[0657] The concentration of 0.15 mg / mL was also studied, which is 1 / 10 of the lowest dose concentration in clinical trials. Changes were observed in protein concentration and ELISA tests. The changes in ELISA tests may be caused by changes in protein concentration, which may be caused by protein adsorption to the contact material. In summary, it was decided to use 5% glucose as the diluent for clinical use.

[0658] In Table 54 below, the bulk analysis data of non-clinical batch 201901004 and clinical batch 201903038 are listed exemplarily.

[0659] Table 54. Bulk analysis

[0660]

[0661]

[0662] 1 Acceptance criteria were not set for non-clinical batches; the reported data are for reference only.

[0663] 2 This result is the average of 6 injections from retesting. The initial result of (LC+HC) was 92.2%. The root cause of the initial result was not determined.

[0664] As a reference standard, a composition containing 50 mg / mL antibody was used, which was formulated in 20 mM histidine buffer, 8% (w / v) sucrose and 0.03% (w / v) PS80 (pH 5.8) and stored in vials filled with 100 μl at -70±10 °C. Additional tests were performed to qualify the reference standard, and the results are shown in Table 55.

[0665] Table 55. Qualitative results of reference standards

[0666]

[0667] The protein concentration and potency of the reference standard were calibrated. The potency of the reference standard was assigned a value of 100% relative potency.

[0668] Stability study

[0669] The non-clinical batch 201901004 and the clinical batch 201903038 were subjected to stability testing. The non-clinical batch (201901004) has 1-month stress stability data, 6-month accelerated stability data, and 18-month long-term stability data. The clinical batch (201903038) has 1-month stress stability data, 6-month accelerated stability data, and 12-month long-term stability data. Under stress conditions, both the clinical batch (201903038) and the non-clinical batch (201901004) showed a trend of significant decrease in the main peak (%) of iCIEF and increase in the acidic peak (%), while no significant changes were observed in other purity assays and ELISA binding assays.

[0670] The shelf life of the drug product is currently set at 24 months when stored in the dark at 5 ± 3°C. The available in-use stability and compatibility data provided above indicate that the ready-to-use solution for infusion is stable for up to 24 hours at 2°C - 8°C after dilution with 5% glucose solution, and then stable for 6 hours at 25°C. From a microbiological perspective, the infusion 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°C - 8°C.

[0671] Tables 56 to 59 summarize the available stress data for the non-clinical batch 201901004 and the clinical batch 201903038.

[0672] Table 56. Stress stability data of non-clinical batch 201901004 at 40±2°C / 75±5% RH

[0673]

[0674]

[0675] Table 57. Stress stability of exploratory specifications of non-clinical batch 201901004 at 40±2°C / 75±5% RH Data

[0676] Testing T0 1 week 2 weeks 1 month Cell-based assay 129％ 121％ 105％ 148％

[0677] Table 58. Stress stability data of clinical batch 201903038 at 40±2°C / 75±5% RH

[0678]

[0679]

[0680] Table 59. Stress stability of exploratory specifications of clinical batch 201903038 at 40±2°C / 75±5% RH Data

[0681] Testing T0 1 week 2 weeks 1 month Cell-based assay 117％ 136％ 111％ 81％

[0682] Tables 60 to 63 summarize the available accelerated data for non-clinical batch 201901004 and clinical batch 201903038.

[0683] Table 60. Accelerated stability data of non-clinical batch 201901004 at 25±2°C / 60±5% RH

[0684]

[0685]

[0686] Table 61. Accelerated stability of exploratory specifications of non-clinical batch 201901004 at 25±2°C / 60±5% RH Data

[0687] Testing T0 1 month 3 months 6 months Cell-based assay 129％ 117％ 140％ 94％

[0688] Table 62. Accelerated stability data of clinical batch 201903038 at 25±2°C / 60±5% RH

[0689]

[0690]

[0691] Table 63. Accelerated stability of exploratory specifications of clinical batch 201903038 at 25±2°C / 60±5% RH Data

[0692] Test T0 1 month 3 months 6 months Cell-based assay 117％ 129％ 108％ 122％

[0693] Tables 64 to 69 summarize the available long-term data for non-clinical batch 201901004 and clinical batch 201903038.

[0694] Table 64. Long-term stability data of non-clinical batch 201901004 at 5±3°C (0 to 9 months)

[0695]

[0696]

[0697] Table 65. Long-term stability data of non-clinical batch 201901004 at 5±3°C (11 to 18 months)

[0698]

[0699]

[0700] Table 66. Long-term stability data of exploratory specifications of non-clinical batch 201901004 at 5±3°C

[0701]

[0702] Table 67. Long-term stability data of clinical batch 201903038 at 5±3°C (0 to 6 months)

[0703]

[0704]

[0705] Table 68. Long-term stability data of clinical batch 201903038 at 5±3°C (9 to 12 months)

[0706]

[0707]

[0708] Table 69. Long-term stability data of exploratory specifications of clinical batch 201903038 at 5±3°C

[0709]

[0710] From the stress stability data, accelerated stability data, and long-term stability data shown in Tables 55 to 69, it can be concluded that the tested pharmaceutical formulation is stable over the long term. For example, the formulation remains liquid without visible particles, the pH remains constant, and the monomer content measured by SEC-HPLC does not drop below 96%, which means that the contents of HMWS and LMWS remain below 4% under all test conditions. In addition, as measured by iCIEF, the amount of acidic substances does not exceed 40% during the long-term stability test, and the ELISA binding assay shows that the antibody retains its binding ability (under all test conditions, it does not drop below 70% of the reference standard, and even does not drop below 95% during the long-term stability study).

[0711] Example 3. Characterization of mature NI006 / ALXN2220

[0712] The antibody NI006 / ALXN2220 is produced in the CHO-K1 cell line with ATCC number CCL 61 and obtained from the 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 shown in SEQ ID NO:9 and 10, with the following modifications. The total number of amino acids of the IgG antibody, the number of amino acids of the heavy chain, and the number of amino acids of the light chain are 1328, 450, and 214, respectively.

[0713] Further characterization of the antibody NI006 / ALXN2022 is mainly carried out by standard procedures such as by mass spectrometry analysis. For example, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of NI006 / ALXN2220 fragments obtained from sequential digestion with Lys-C and trypsin, as well as free thiol analysis, are used to identify the post-translational modifications of NI006 / ALXN2220. Characterizing antibody-based therapeutics via 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.

[0714] N - glycan profiling was performed by releasing N - glycans using PNGase F, followed by labeling with 2 - AB and then separation by hydrophilic interaction chromatography (HILIC) and fluorescence detection (FLD) using a UPLC system. Individual N - glycans and unknown peaks were quantified by their percentage of peak area relative to the total peak area.

[0715] The results are as follows :

[0716] The molecular weights of the antibody NI006 / ALXN2220 determined by standard mass spectrometry are approximately 147.1 kDa for the intact IgG1 and 144.2 kDa for the deglycosylated variant.

[0717] The monoclonal antibody NI006 / ALXN2220 is an IgG1 subclass antibody consisting of two heavy chains of the IgG1 subclass and two light chains of the κ subclass. The four chains are stabilized by multiple disulfide bonds. Specifically, as determined by standard procedures, i.e., based on Lys - C and trypsin digestions and subsequent LC - MS, the following disulfide bridges are present in NI006 / ALXN2220:

[0718] LC:C23 - LC:C88

[0719] LC:C134 - LC:C194

[0720] LC:C214 - HC:C223

[0721] HC:C22 - HC:C97

[0722] HC:C147 - HC:C203

[0723] HC1:229 - HC2:229 and HC1:232 - HC2:232

[0724] HC:C264 - HC:C324

[0725] HC:C370 - HC:C428

[0726] (The corresponding amino acid sequences of HC and LC are shown in SEQ ID NO:9 and 10)

[0727] NI006 / ALXN2220 is a glycoprotein, and the constant region of each heavy chain contains an N - linked glycan site at residue N300. During glycan profiling, the major N - glycan types were shown to be G0F (approx. 49.0%) and G1F (approx. 25.4%). More specifically, the following glycan characteristics (type of sugar, position of glycosylation site, etc.) of NI006 / ALXN2220 were determined:

[0728] Table 70: Glycan type

[0729] Glycan Glycan type 23100 Man3+1F 33000 G0-GN 33100 G0F-GN 43000 G0 43100 G0F 25000 Man5 34100 / 44000 G1F-GN / G1a 44000 G1b 44100 G1Fa 44100 G1Fb 45100 G2F 45110 G2FS1 45120 G2FS2

[0730] Note:

[0731] 1. The nomenclature of the glycan follows the order of HexNac - hexose - fucose - NeuAc - NeuGc. For example, 23000 is HexNac(2)-hexose(3)-fucose(0)-NeuAc(0)-NeuGc(0).

[0732] 2. G1Fa and G1Fb are isomers and are grouped as G1F. G1F is calculated as the sum of G1Fa and G1Fb using the raw unrounded numbers.

[0733] In addition, the glutamine at the N-terminus of the heavy chain is modified to pyroglutamic acid (abundance in the sample: 99.9%) and the cleavage of the C-terminal lysine (abundance in the sample: 95.8%) have been identified as the major post-translational modifications. In addition, as shown in Table 71, minor modifications such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation have been experimentally determined:

[0734] Table 71: Heavy chain partial sequence

[0735]

[0736]

[0737] Note:

[0738] 1. HC refers to the heavy chain and LC refers to the light chain.

[0739] 2. The underlined peptide sequences are identified as the sites of PTM.

[0740] 3. * refers to the N-terminal related peptide of the heavy chain and # refers to the C-terminal related peptide of the heavy chain.

[0741] 4. / refers to the unreported PTM.

[0742] 5. pE(Q) refers to the N-terminal glutamine modified to pyroglutamic acid.

[0743] 6. -K refers to the deletion of the C-terminal lysine.

[0744] 7. -K-G amidation(P) refers to the amidation of the C-terminal proline after the deletion of the C-terminal lysine and glycine.

[0745] In summary, the N-linked glycosylation of the heavy chain, the N-terminal pyroglutamic acid modified from the N-terminal glutamine, and the cleavage of the C-terminal lysine of the heavy chain are the major post-translational modifications of NI006 / ALXN2220

[0746] Other embodiments

[0747] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference

[0748] Although the invention has been described in connection with specific embodiments thereof, it is to be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention that generally follow the principles of the invention and include such departures therefrom as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features set forth above and fall within the scope of the claims

Claims

1. A pharmaceutical composition, the pharmaceutical composition comprising a human anti-TTR antibody or an antigen-binding fragment thereof that is capable of binding to aggregated transthyretin (TTR) substance and substantially does not recognize physiological TTR substance, wherein the pharmaceutical composition comprises one or more of sucrose, polysorbate 80, and a polar excipient.

2. The pharmaceutical composition according to claim 1, the pharmaceutical composition being an aqueous formulation.

3. The pharmaceutical composition according to claim 1 or 2, wherein the antibody binds to mutant and wild-type aggregated TTR substances.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody does not bind to monomers and dimers of human native TTR.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antibody binds to a TTR epitope comprising the amino acid sequence EEEFVEGIY (SEQ ID NO:49), GELHGLTTEEE (SEQ ID NO:50), or WEPFA (SEQ ID NO:51) or consisting thereof.

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) sucrose.

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

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

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

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

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

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

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

3.

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

9.

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

8.

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

5.

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

1.

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

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

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein the polar excipient comprises about 20 mM of 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. The pharmaceutical composition according to any one of claims 1 to 22, wherein the anti-TTR antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) region having three complementarity-determining regions (CDRs) shown in SEQ ID NOs: 1-3 and a light chain variable (VL) region having three CDRs shown in SEQ ID NOs: 4-6.

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

8.

25. The pharmaceutical composition according to 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 acids of SEQ ID NO:

8.

26. The pharmaceutical composition according to 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 acids of SEQ ID NO:

12.

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

28. The pharmaceutical composition according to 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 a human IgG1 type.

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

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

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

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

10.

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

10.

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

10.

36. The pharmaceutical composition according to any one of claims 1 to 32, wherein the antibody comprises two heavy chains and two light chains, wherein each heavy chain consists of 448 amino acid residues having SEQ ID NO: 15, and each light chain consists 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 a CHO host cell, preferably in a CHO-K1 cell, and purified from the cell culture.

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

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

40. The antibody for use in a method according to any one of claims 27 to 39, wherein the antibody is N-glycosylated, preferably wherein the N-linked glycan is located at N300 of the heavy chain.

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

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

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

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

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

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

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

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

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

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

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

52. The pharmaceutical composition according to any one of claims 1 to 51, wherein the pharmaceutical composition is characterized by any combination of one, two, three, or all four stability criteria (a) to (d): (a) The main peak decrease under heat stress conditions of 4 weeks at 40 °C and / or 12 weeks at about 25 °C is less than 1% by weight of the antibody, as measured by size exclusion chromatography (SEC)- HPLC analysis; (b) The pharmaceutical composition shows that the content of acidic substances of the anti-TTR antibody or an antigen-binding fragment thereof under heat stress conditions of 2 weeks at about 40 °C is less than 42.5%, as measured by capillary isoelectric focusing (cIEF); (c) After 3 cycles of freeze-thaw (-70 °C to room temperature (RT)), the pharmaceutical composition shows that the content of acidic substances of the anti-TTR antibody does not change significantly, as measured by capillary isoelectric focusing (cIEF); and / or (d) The anti-TTR antibody or an antigen-binding fragment thereof retains at least 80% of the binding efficacy to TTR protein after storage at 40 °C for 4 weeks and / or retains at least 70% of the binding efficacy to TTR protein after storage at 25 °C for 12 weeks, as measured by ELISA and relative to a control.

53. The pharmaceutical composition according to any one of claims 1 to 52, wherein the pharmaceutical composition shows a main peak ≥ 50.0%, an acidic peak ≤ 40.0%, and a basic peak ≤ 15.0% as measured by, for example, cIEF, a main peak (monomer) ≥ 95.0% and high molecular weight substances (HMWS) ≤ 5.0% as measured by, for example, size exclusion chromatography (SEC)-HPLC analysis, pH 5.8 ± 0.5, osmolarity ≥ 240 mOsm / Kg, and an antibody concentration of 50 ± 5.0 mg / mL.

54. The pharmaceutical composition according to any one of claims 1 to 53, wherein the pharmaceutical composition is characterized by any combination of one, two, three, or all four stability criteria (i) to (iv): (i) The main peak decrease is 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 shows that the content of acidic substances of the anti-TTR antibody or its antigen-binding fragment is less than or approximately equal to about 40% 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 shows that the content of acidic substances of the anti-TTR antibody or its antigen-binding fragment is less than 40%, preferably less than 35%, as measured by cIEF, under long-term storage conditions at about 5 °C for 12 months or 18 months; and / or (iv) The anti-TTR antibody or its antigen-binding fragment retains at least 80%, preferably at least 90%, of its binding potency to the TTR protein after storage at about 25 °C for 6 months or after storage at about 5 °C for 12 months or 18 months, as measured by ELISA and relative to a control.

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

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

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

58. The pharmaceutical composition according to any one of claims 1 to 57, wherein the pharmaceutical composition is present in one or more vials or infusion bottles, and the vials or infusion bottles in total contain from about 2500 mg to about 7500 mg of the anti-TTR antibody or its antigen-binding fragment.

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 having a volume of 2 mL to 20 mL, such as a 2 mL or 20 mL type I clear glass vial.

60. The pharmaceutical composition according to claim 59, wherein the vial or infusion bottle contains an approximate 12.5% volume overfill, or a total volume of approximately 2.25 mL or 22.5 mL of the anti-TTR antibody or its antigen-binding fragment.

61. The pharmaceutical composition according to any one of claims 1 to 60, wherein: (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 (L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), a pH of 5.8, and a volume of 2 mL to 50 mL; and (b) The anti-TTR antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:7 and a VL region having the amino acid sequence of SEQ ID NO:

8.

62. The pharmaceutical composition according to any one of claims 1 to 60, wherein: (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 (L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), a pH of 5.8, and a volume of 2 mL to 50 mL; and (b) The anti-TTR antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:7 and a VL region having the amino acid sequence of SEQ ID NO:

8.

63. The pharmaceutical composition according to any one of claims 1 to 60, wherein: (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 (L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), a pH of 5.8, and a volume of 2 mL to 50 mL; and (b) The anti-TTR antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:11 and a VL region having the amino acid sequence of SEQ ID NO:

12.

64. The pharmaceutical composition according to any one of claims 1 to 60, wherein: (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 (L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), a pH of 5.8, and a volume of 2 mL to 50 mL; and (b) The anti-TTR antibody or antigen-binding fragment thereof comprises a VH region having the amino acid sequence of SEQ ID NO:11 and a VL region having the amino acid sequence of SEQ ID NO:

12.

65. A pharmaceutical composition, the pharmaceutical composition comprising a human anti-transthyretin (TTR) antibody or an 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; wherein the anti-TTR antibody or an antigen-binding fragment thereof is capable of binding to mutant, misfolded, misassembled, and / or aggregated TTR substance and / or a fragment thereof, and substantially does not recognize physiological TTR substance.

66. A pharmaceutical composition, the pharmaceutical composition being formulated for intravenous injection, the pharmaceutical composition comprising 50 mg / mL of a human anti-transthyretin (TTR) antibody, the 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 / 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 a pH of 5.

8.

67. The pharmaceutical composition according to 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. The pharmaceutical composition according to 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. The pharmaceutical composition according to 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. The pharmaceutical composition according to any one of claims 66 to 69, wherein the pharmaceutical composition has a volume of 2 mL.

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

72. A pharmaceutical composition, the pharmaceutical composition being formulated for intravenous injection, the pharmaceutical composition comprising 50 mg / mL of a human anti-TTR antibody, the human anti-TTR 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 (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 a pH of 5.

8.

73. The pharmaceutical composition according to 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. The pharmaceutical composition according to 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. The pharmaceutical composition according to 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. The pharmaceutical composition according to any one of claims 72 to 75, wherein the pharmaceutical composition has a volume of 2 mL.

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

78. A pharmaceutical composition, the pharmaceutical composition being formulated for intravenous injection, the pharmaceutical composition comprising approximately 50 mg / mL of a human anti-transthyretin (TTR) antibody, the human anti-transthyretin (TTR) antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO: 9 and each light chain has the amino acid sequence of SEQ ID NO: 10, wherein the pharmaceutical composition comprises 6.5% weight / 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 a pH of 5.

8.

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

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

15.

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

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

83. A pharmaceutical composition, which is formulated for intravenous injection, the pharmaceutical composition comprising about 50 mg / mL of a human anti-transthyretin (TTR) antibody, the human anti-transthyretin (TTR) antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO:9, and each light chain has the amino acid sequence of SEQ ID NO:10, wherein the pharmaceutical composition comprises 8% weight / 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 a pH of 5.

8.

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

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

15.

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

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

88. A kit, which comprises the pharmaceutical composition according to any one of claims 1 to 87 and instructions for use thereof.

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

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

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

92. A method for treating transthyretin-mediated amyloidosis (ATTR) in a human subject, the method comprising administering a pharmaceutical composition comprising 50 mg / ml of a human anti-TTR antibody, the human anti-TTR antibody comprising 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, wherein the pharmaceutical composition comprises 6.5% w / v sucrose, 0.03% w / v polysorbate 80, 20 mM histidine, and a pH of 5.

8.

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

97. A method of treating ATTR in a human subject, the method comprising administering a pharmaceutical composition comprising a human anti-TTR antibody at 50 mg / ml, the human anti-TTR antibody comprising 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, 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. The method according to 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. The method according to 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. The method according to 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. The method according to any one of claims 97 to 100, wherein the method comprises administering to the subject a dose of the pharmaceutical composition that provides 30 mg / kg to 60 mg / kg of the human anti-TTR antibody or at most 7500 mg of the human anti-TTR antibody.

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

8.

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

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

15.

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

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

8.

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

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

15.

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

110. A medicament comprising the pharmaceutical composition according to any one of claims 1 to 87 for use in a method of treating ATTR in a subject in need thereof, preferably for use in treating a subject suffering from ATTR amyloid cardiomyopathy (ATTR-CM).

111. A sterile pharmaceutical container comprising the pharmaceutical composition according to any one of claims 1 to 87 or the medicament according to claim 110, preferably wherein the container is a disposable glass vial preferably sealed with a rubber stopper and an aluminum-plastic cap flip-top, or an infusion bag, or a metering syringe in a metering pump, which contains about 100 mg of the antibody at a concentration of about 50 ± 5 mg / mL to about 150 mg / mL, such as a concentration of about 100 mg / mL, optionally wherein the vial contains an approximate 10% volume overfill.

112. The medicament according to claim 110 or the container according to claim 111, wherein the pharmaceutical composition is ready-to-use for administration to a subject in need thereof, preferably via intravenous infusion.

113. The medicament or container according to claim 112, wherein the pharmaceutical composition is diluted in a solution of glucose or its polymer before infusion, preferably wherein the polymer is dextran.

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

115. The medicament or container according to claim 113 or 114, wherein the concentration of the glucose or its polymer is 5% w / v.

116. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of ATTR in a subject, wherein the pharmaceutical composition comprises: (a) a human anti-TTR antibody or an antigen-binding fragment thereof that is capable of binding to aggregated TTR material and substantially does not recognize physiological TTR material, and (b) one or more of sucrose, polysorbate 80, and a polar excipient.

117. The use according to claim 116, wherein the human anti-TTR antibody or an antigen-binding fragment thereof is present in the medicament at a concentration of about 50 mg / mL to about 150 mg / mL, and wherein the medicament comprises a histidine buffer with 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 the pharmaceutical composition according to any one of claims 1 to 87.

119. Use of a pharmaceutical composition in the manufacture of a medicament for the treatment of ATTR in a human subject, wherein the pharmaceutical composition comprises about 50 mg / ml of a human anti-TTR antibody, the human anti-TTR 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; and 20 mM histidine and a pH of 5.

8.

120. The use according to 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 according to 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 according to 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. Use of a pharmaceutical composition in the preparation of a medicament for the treatment of ATTR in a human subject, wherein the pharmaceutical composition comprises a human anti-TTR antibody at about 50 mg / ml, the human anti-TTR 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; and 20 mM histidine and a pH of 5.

8.

124. The use according to 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 according to 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 according to 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. Use of a pharmaceutical composition in the preparation of a medicament for the treatment of ATTR in a human subject, wherein the pharmaceutical composition comprises a human anti-TTR antibody at 50 mg / ml, the human anti-TTR antibody comprising two heavy chains and two light chains, wherein each heavy chain has the amino acid sequence of SEQ ID NO: 9 and each light chain has the amino acid sequence of SEQ ID NO: 10; wherein the pharmaceutical composition comprises 6.5% w / v sucrose; 0.03% w / v polysorbate 80; and 20 mM histidine and a pH of 5.

8.

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

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

15.

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

8.

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

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

15.

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

134. The article according to claim 133, wherein the formulation of the antibody is provided as a concentrate of a solution for infusion and is preferably presented as a preservative-free, clear to milky white and colorless to light yellow solution, provided in vials at 100 mg / 2 mL.

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

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

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