Anti-transthyretin antibody, a composition comprising the antibody, and a method for treating or preventing transthyretin-mediated amyloidosis

Abstract

Anti-transthyretin (TTR) antibodies, corresponding polynucleotides, and expression vectors, as well as compositions (e.g., pharmaceutical compositions) containing anti-TTR antibodies as drugs and related manufactured articles are provided herein. Methods for treating or preventing transthyretin-mediated amyloidosis (ATTR) in subjects requiring such treatment or prevention using the pharmaceutical compositions described herein are also provided herein. Additionally, methods for verifying, identifying, and screening amyloid-depleting drugs using high-resolution live cell imaging are provided herein, as well as methods for producing pharmaceutical compositions of amyloid-depleting drugs and kits suitable for use in said methods.

Description

Technology Field

[0001] The present disclosure relates to an anti-transthyretin (TTR) antibody, a corresponding polynucleotide, and an expression vector, as well as a composition comprising said antibody and a method for treating or preventing transthyretin-mediated amyloidosis (ATTR). Additionally, the present disclosure relates to a method for verifying, identifying, and screening amyloid-depleting drugs using high-resolution live cell imaging, as well as a method for producing a pharmaceutical composition of an amyloid-depleting drug and a kit suitable for use in said method. Background Technology

[0002] Transthyretin (TTR) is a soluble protein involved in the transport of thyroxine and retinol in the body. TTR is secreted into the blood by the liver and into the cerebrospinal fluid by the choroid retina, and is also expressed in certain tissues such as pancreatic alpha cells or retinal epithelium.

[0003] Under specific, yet-unclear conditions that may include acidic pH, oxidative stress, and local factors, TTR proteins may misfold / misassemble / or aggregate TTR structures, become toxic, and lead to transthyretin-mediated amyloidosis (ATTR).

[0004] There is a need for antibodies targeting aggregated TTR (e.g., human anti-TTR antibodies) suitable for treating or preventing ATTR in subjects, and for drug products for administering the antibodies.

[0005] In particular, the present specification provides anti-transthyretin (TTR) antibodies, corresponding polynucleotides and expression vectors, as well as compositions (e.g., pharmaceutical compositions) containing anti-TTR antibodies as drugs and related manufactured articles. Additionally, the present specification provides a method for treating or preventing transthyretin-mediated amyloidosis (ATTR) using the pharmaceutical compositions described herein, particularly in subjects requiring treatment or prevention.

[0006] According to the present disclosure, a human anti-TTR antibody specific to the disease-associated amyloidogenic form of TTR, characterized by ALXN2220, also known as NI006 and designated by CAS registration number 2965214-50-8, was prepared by recombinantly expressing the heavy chain (HC) and light chain (LC) of the antibody in Chinese hamster ovary (CHO) K1 cell lines, and this recombinant antibody possesses a unique post-translational modification (PTM) pattern, in particular a unique glycosylation profile; see Examples 1 to 3.

[0007] The antibody NI006 / ALXN2220 is a fully human IgG1m3 allogeneic antibody and comprises a human constant heavy chain (HC) amino acid sequence as presented for SEQ ID NOs 7, 9, and 39, respectively, and a corresponding human constant light chain (LC) (here, kappa light chain) as presented for SEQ ID NO. 8. As further described below, the NI006 / ALXN2220 IgG1 antibody consists of a tetramer composed of HC and two light chain LCs linked by disulfide crosslinks. Furthermore, the closest human genes / alleles of the variable domain are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).

[0008] The antibody CDR of NI006 / ALXN2220 (e.g., variable heavy chain (VH) and / or variable light chain (VL) CDR comprising VHCDR1 to 3 and VLCDR1 to 3; the same includes variable heavy chain and light chain domains) is WO 2015 / 092077 A1 (designated as antibody NI-301.37F1) and literature [Michalon et al It is first described in [., Nat. Commun. 12 (2021), 3142] (designated as antibody NI301A), the disclosures of which are incorporated herein by reference in the relevant parts. The complete (e.g., full-length) variable heavy chain (VH) and light chain (VL) of the NI006 / ALXN2220 antibody—including the heavy chain and / or light chain constant regions—are described herein. The present disclosure further provides a full-length NI006 / ALXN2220 antibody comprising a PTM in the heavy chain and / or light chain, which is particularly suitable for providing a stable formulation that can be used as a pharmaceutical composition in the treatment of ATTR.

[0009] In particular, the antibody of the present disclosure comprises a modified glutamine at the N-terminus of the heavy chain sequence, wherein the modification comprises pyroglutamate (the abundance in the sample, e.g., a recombinant formulation of the antibody is about 99.9% or greater, e.g., 100%). In further embodiments, the antibody of the present disclosure comprises a clipped C-terminal lysine in the heavy chain (the abundance in the sample, e.g., a recombinant formulation of the antibody is about 95.8% or greater, e.g., 96%, 97%, 98%, 99%, or even 100%). The NI006 / ALXN2220 antibody subtype was identified by antibody heavy chain sequence analysis to contain both N-terminal pyroglutamate and C-terminal lysine deletions as major post-translational modifications.

[0010] N-terminal pyroglutamate modification is beneficial to therapeutic antibodies by improving stability and consistency and reducing immunogenicity. In particular, N-terminal pyroglutamate modification helps protect the antibody's N-terminus from enzymatic proteolytic degradation, which increases antibody stability and extends the antibody's half-life in circulation. Furthermore, pyroglutamate formation improves the homogeneity of antibody formulations by ensuring a consistent N-terminal structure throughout the molecule. This consistency produces more uniform products with predictable characteristics, which can be advantageous for quality control and regulatory approval. Moreover, it helps reduce the risk of immunogenicity. By cleaving N-terminal glutamine or glutamate, it prevents unwanted immune responses that could be triggered by unmodified N-terminal residues that would otherwise be recognized as foreign substances. The enzyme responsible for converting N-terminal glutamine (or sometimes glutamate) to pyroglutamate is called glutaminyl cyclase. The activity level of this enzyme may vary depending on the cell type and can have a significant effect on the degree of pyroglutamate formation.

[0011] C-terminal lysine clipping is advantageous because it improves charge homogeneity, reduces immunogenicity, enhances consistency, and does not affect antibody functionality. In particular, the presence of C-terminal lysine can introduce charge heterogeneity across the antibody population by adding a positive charge to the antibody. By clipping C-terminal lysine, antibody formulations become more charge-uniform, simplifying downstream processes such as purification, and improving batch-to-batch consistency and homogeneity, which is beneficial for quality control. Furthermore, antibodies in vivo ( in vivoC-terminal lysine is naturally deficient due to proteolytic elimination. If lysine remains, the antibody may be recognized by the immune system as a foreign substance, potentially triggering an immune response. Removing this lysine reduces the risk of immunogenicity, making therapeutic antibodies more biocompatible. Since C-terminal lysine residues do not play a role in the antibody binding to its target antigen or interacting with Fc receptors, their removal does not affect the antibody's therapeutic activity; therefore, cleaving them is beneficial without compromising efficacy. The efficiency of C-terminal lysine clipping may vary depending on the cell line used for antibody production, as different cell lines differ in their protease activity and efficiency in removing C-terminal lysine residues from antibodies.

[0012] Therefore, an antibody having N-terminal pyroglutamate modification and C-terminal lysine clipping is beneficial for therapeutic purposes and is thus a preferred embodiment of the present invention.

[0013] Furthermore, additional modifications such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation were experimentally determined as presented in Table 3. Accordingly, the present disclosure relates to SEQ ID NO. 7 (wherein,

[0014] X1 is absent, glutamine, or pyroglutamate (pE);

[0015] X2 is methionine or oxidized methionine;

[0016] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide;

[0017] X4 is asparagine or deamidated asparagine;

[0018] X5 is proline or amidated proline;

[0019] X6 is absent or glycine;

[0020] HC having an amino acid sequence of X7 (absent or lysine),

[0021] The invention relates to a mature anti-TTR antibody comprising LC having the amino acid sequence of sequence number 8.

[0022] Accordingly, in one embodiment, the antibody of the present disclosure comprises HC having the amino acid sequence of SEQ ID NO. 7, wherein the N-terminal glutamine (Q1) denoted as X1 in SEQ ID NO. 7 is absent or present, preferably absent. In an embodiment, X1 is present as glutamine or as pyroglutamate (pE); preferably, X1 is pE.

[0023] Additionally or alternatively, the antibody of the present disclosure comprises HC having the amino acid sequence of SEQ ID NO. 7, wherein the C-terminal lysine (K450) denoted as X7 in SEQ ID NO. 7 is absent or present. In a preferred embodiment, X7 is absent.

[0024] In one embodiment, the antibody of the present disclosure comprises HC having the amino acid sequence of SEQ No. 39, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X I is asparagine or deamidated asparagine. In an embodiment, X II is methionine or oxidized methionine. In an embodiment, X III is methionine or oxidized methionine. In an embodiment, X IVis aspartate or iso-aspartate. In an embodiment, X V is asparagine or glycosylated asparagine. In an embodiment, X VI is methionine or oxidized methionine. In an embodiment, X VII It is methionine or oxidized methionine. In an embodiment, the antibody comprises LC having the amino acid sequence of SEQ ID NO. 8.

[0025] In an embodiment, the antibody of the present disclosure comprises HC having the amino acid sequences for SEQ ID NOs. 7 and 39, respectively. In some embodiments, X1 is pE. In some embodiments, X6 is absent. In some embodiments, X7 is absent. In some embodiments, X5 is amidated. In some embodiments, X2 is oxidized methionine. In some embodiments, X3 is deamidated asparagine. In other embodiments, X3 is asparagine containing succinimide. In some embodiments, X4 is deamidated asparagine.

[0026] In some embodiments, X I is deamidated asparagine. In some embodiments, X II is oxidized methionine. In some embodiments, X III is oxidized methionine. In some embodiments, X IV is iso-aspartate. In some embodiments, X V is glycosylated asparagine. In some embodiments, X VI is oxidized methionine. In some embodiments, X VII It is oxidized methionine.

[0027] In some embodiments, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, and X7 is absent.

[0028] In some preferred embodiments, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X I is asparagine, and X II is methionine, and X III is methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII It is methionine.

[0029] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is proline, X6 is glycine, and X7 is absent.

[0030] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is amidated proline, X6 is absent, and X7 is absent.

[0031] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is proline, X6 is glycine, X7 is absent, and X I is asparagine, and X II is methionine, and X III is methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII It is methionine.

[0032] In some embodiments, X1 is pE, X2 is oxidized methionine, X3 is deamidated asparagine or asparagine containing succinimide, X4 is deamidated asparagine or asparagine containing succinimide, X5 is amidated proline, X6 is absent, X7 is absent, and X I is asparagine, and X II is methionine, and X III is methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII It is methionine.

[0033] In an embodiment, the C-terminal lysine (C450, numbering based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) of the antibody of the present disclosure may be clipped (e.g., through proteolytic cleavage). Accordingly, in an embodiment, the antibody of the present disclosure comprises a HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein the C-terminal lysine designated as X7 for each of SEQ ID NOs 7 and 39 is absent or present. Preferably, the C-terminal lysine in X7 for each of SEQ ID NOs 7 and 39 is absent.

[0034] In an embodiment of the antibody of the present disclosure in which C-terminal lysine is absent in the HC, the resulting C-terminal glycine (G449, numbering based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) may also be modified. Accordingly, in an embodiment, the antibody of the present disclosure comprises a HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein the C-terminal lysine at X7 is absent, and for each of SEQ ID NOs 7 and 39, the adjacent amino acid, denoted as glycine at X6 at their C-terminus, is absent or present.

[0035] In some embodiments of the antibody of the present disclosure in which both C-terminal lysine and glycine are absent in the HC, the resulting C-terminal proline (P448, numbering based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) may be amidated. Accordingly, in one embodiment, the antibody of the present disclosure comprises a HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein both C-terminal lysine at X7 and glycine at X6 are absent, and for each of SEQ ID NOs 7 and 39, the adjacent amino acid, denoted as proline at X5 at their C-terminus, is amidated or unmodified, preferably amidated.

[0036] Additionally or alternatively, in an embodiment, methionine at position 255 (M255, numbering based on an HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) in the heavy chain of the antibody of the present disclosure may be oxidized. Thus, in one embodiment, the antibody of the present disclosure comprises an HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein M255, denoted as methionine at X2 for each of SEQ ID NOs 7 and 39, is oxidized (modified) or unmodified.

[0037] Additionally or alternatively, in an embodiment, the asparagine at position 318 (N318, numbering based on an HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) in the heavy chain of the antibody of the present disclosure may be unmodified or deamidated or may contain succinimide. Accordingly, in one embodiment, the antibody of the present disclosure comprises an HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein N318, denoted as the asparagine in X3 for each of SEQ ID NOs 7 and 39, is modified or unmodified, and preferably, the modified asparagine in X3 is deamidated (modified) or contains succinimide (modified).

[0038] Additionally or alternatively, in an embodiment, the asparagine at position 387 (N387, numbering based on the HC polypeptide sequence containing N-terminal glutamine or pyroglutamate) in the heavy chain of the antibody of the present disclosure may be unmodified or modified, for example, the modified amino acid is deamidated. Accordingly, in one embodiment, the antibody of the present disclosure comprises HC having the amino acid sequences for each of SEQ ID NOs 7 and 39, wherein N387, denoted as the asparagine at X4 for each of SEQ ID NOs 7 and 39, is deamidated (modified) or unmodified.

[0039] In an embodiment, the antibody of the present disclosure comprises N-glycosylation. In some embodiments, at least one amino acid in the heavy chain of the antibody of the present disclosure is N-glycosylated. The data of Example 2 show that N-glycosylation is a substantial PTM of the backbone amino acid sequence constituting the antibody of the present disclosure. In this regard, the N-glycosylation site was identified at position Asn300 (HC N300, numbering based on the antibody HC polypeptide sequences for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1).

[0040] In some preferred embodiments, the present disclosure relates to an antibody comprising the heavy chain polypeptide sequences presented for SEQ ID NOs. 7 and 39, respectively, wherein X1 is pyroglutamate, X7 is cleaved, and asparagine (N) at amino acid position 300 is N-glycosylated.

[0041] In some preferred embodiments, the present disclosure relates to an anti-TTR antibody comprising an HC comprising the sequences presented for SEQ ID NOs. 7 and 39, respectively, wherein glutamine (X1) is modified to pyroglutamate, lysine (X7) is absent, the amino acids at positions X2, X3, X4, X5, and X6 are modified or unmodified as indicated above, preferably unmodified, and at least one amino acid is N-glycosylated. Preferably, in such embodiments, the N-glycosylated site is located at position Asn300 (HC N300, numbering based on the antibody HC polypeptide sequences for SEQ ID NOs. 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1).

[0042] In a specific embodiment, the antibody of the present disclosure comprises pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, wherein the abundance in the sample is about 99.9% or more, e.g., 100%. In a further specific embodiment, the antibody of the present disclosure comprises a C-terminus in which C-terminal lysine in the heavy chain is clipped, wherein the abundance in the sample is about 95.8% or more, e.g., 96%, 97%, 98%, 99%, or even 100%. In a further specific embodiment, the antibody of the present disclosure comprises deamidated asparagine at position 58 (N58 is based on the antibody HC polypeptide sequence for SEQ NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises deamidated asparagine at position 318 (N318 is based on the antibody HC polypeptide sequence for SEQ NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 9% or less, e.g. 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises deamidated asparagine at position 387 (N387 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises oxidized methionine at position 71 (M71 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%.In a further specific embodiment, the antibody of the present disclosure comprises oxidized methionine at position 115 (M15 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises oxidized methionine at position 255 (M255 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises oxidized methionine at position 361 (M361 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. In a further specific embodiment, the antibody of the present disclosure comprises oxidized methionine at position 431 (M413 is based on the antibody HC polypeptide sequence for SEQ ID NOs 7 and 39, respectively, having N-terminal glutamine or pyroglutamate (X1)), wherein the abundance in the sample is about 5% or less, e.g. 4%, 3%, 2%, 1%, or 0%. Furthermore, the antibody of the present disclosure may comprise a glycosylated heavy chain and / or a glycosylated light chain.

[0043] As mentioned above, antibodies having N-terminal pyroglutamate modification and C-terminal lysine clipping are beneficial for therapeutic purposes, but it is preferable that the abundance of the other PTMs mentioned be rather low. For example, methionine oxidation, iso-aspartate formation, or deamidated asparagine in therapeutic antibodies are generally not advantageous because they can have a negative effect on stability, function, safety, and efficiency; therefore, the amount of methionine oxidation, iso-aspartate formation, and asparagine deamidation should preferably be about 5% or less.

[0044] Accordingly, in a preferred embodiment, X1 is pE, X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X I is asparagine, and X II is methionine, and X III is methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII It is methionine.

[0045] In an embodiment, the antibody of the present disclosure comprises two HCs having the amino acid sequences presented for SEQ ID NOs 7 and 39, respectively, and two LCs having the amino acid sequences presented for SEQ ID NOs 8, respectively, wherein glutamine (X1) located at the N-terminus of the HC sequence is independently modified to pyroglutamic acid (pE) in each case. In an embodiment, the antibody of the present disclosure comprises two HCs having the amino acid sequences presented for SEQ ID NOs 7 and 39, respectively, wherein the C-terminal lysine (X7) of the HC sequence is independently absent in each case. In an embodiment, the antibody of the present disclosure comprises two HCs having the amino acid sequences presented for SEQ ID NOs 7 and 39, respectively, wherein the heavy chain is independently N-glycosylated in each case, and preferably the N-glycosylated site is located at position Asn300. In an embodiment, the antibody of the present disclosure comprises two HCs comprising the amino acid sequence presented in SEQ ID NO. 7, wherein the amino acids at positions X2, X3, X4, and X5 for each of SEQ ID NO. 7 and 39 are modified or unmodified, preferably unmodified.

[0046] In an embodiment, the antibody of the present disclosure comprises two HCs having the amino acid sequences presented for each of SEQ ID NO. 7 and 39, and two LCs having the amino acid sequences presented for each of SEQ ID NO. 8, wherein glutamine (X1) located at the N-terminus of the HC sequence is independently modified to pyroglutamate (pE) in each case; C-terminus lysine (X7) of the HC sequence is independently absent in each case; the heavy chain is independently N-glycosylated in each case, preferably the N-glycosylated site is present at position Asn300 for each of SEQ ID NO. 7 and 39, and the numbering of Asn corresponds to the polypeptide sequence for each of SEQ ID NO. 7 and 39, comprising glutamine or pyroglutamate at X1; and the amino acids at positions X2, X3, X4, and X5 for each of SEQ ID NO. 7 and 39 are modified or unmodified, preferably unmodified.

[0047] In an embodiment of the present disclosure, the antibody of the present disclosure comprises two HCs and two LCs, wherein the heavy chains each independently have the amino acid sequence presented in SEQ ID NO. 9 and the light chains each independently have the amino acid sequence presented in SEQ ID NO. 8.

[0048] In a preferred embodiment, the heavy chain of the anti-TTR antibody of the present disclosure does not have C-terminal lysine as presented in SEQ ID NO. 9, and the N-terminal glutamine as presented in SEQ ID NO. 9 is modified to pyroglutamate. The sequence of the heavy chain having clipped C-terminal lysine and cyclized N-terminal glutamine (e.g., cyclized modification of glutamine to pyroglutamate) is presented in SEQ ID NO. 12.

[0049] In some embodiments, the antibody of the present disclosure may have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimidation, pyroglutamate formation, and isomerization. A plurality of such PTMs may be present in any combination. In some embodiments, the PTMs are within the heavy chain polypeptide sequence; however, the light chain polypeptide amino acid sequence may also be post-translated modified. PTMs experimentally determined in NI006 / ALXN2220 are provided in detail in Example 2. In particular, in addition to the C-terminal lysine clipping and cyclization from N-terminal glutamine to pyroglutamate mentioned above, the heavy chain and / or light chain of the antibody may undergo, for example, oxidation of methionine (M) at position HC 255; for example, deamidation of asparagine (N) at position HC 318 and / or HC 387; For example, it may contain the formation of asparagine (N) succinimide at position HC 318; and / or the amidation of C-terminal proline (P) after the loss of C-terminal lysine and glycine.

[0050] Additionally or alternatively, the antibody is glycosylated, particularly N-glycosylated. More particularly, the heavy chain of the antibody is glycosylated, and even more particularly, the N300 of the heavy chain (sequence number 9) is glycosylated.

[0051] In a preferred embodiment, the HC chain of the anti-TTR antibody of the present disclosure lacks C-terminal lysine, has glutamine modified as pyroglutamic acid at the N-terminus, and includes at least one N-glycosylation site. Accordingly, in one preferred embodiment, the antibody of the present disclosure consists of two heavy chains having SEQ ID NO. 9 and two light chains having SEQ ID NO. 8, wherein the glutamine at the N-terminus of the heavy chain is modified as pyroglutamic acid, the C-terminal lysine is removed or absent, and the heavy chain is N-glycosylated. In other words, the antibody consists of two heavy chains having sequence number 12 (preferably the N-terminus of each antibody HC is cyclized) and two light chains having sequence number 8, each heavy chain being N-glycosylated, for example, in Asn300. In an additionally preferred embodiment, the antibody has the remaining other PTM in low abundance (preferably about 5% or less as further mentioned above).

[0052] Such antibodies are stable, safe, efficient, have a long half-life, reduced immunogenicity, and exhibit charge homogeneity, making them particularly advantageous for therapeutic purposes. These advantageous features are confirmed in Examples 3 and 4. In particular, the stability study in Example 4 demonstrates that the antibody formulation is stable over a long period, and the batch analysis in Example 3 also confirmed the stability of the antibody. For example, SEC analysis confirms that there are almost no high or low molecular weight species present in the sample (no aggregation or degradation).

[0053] In one embodiment of the present disclosure, the antibody comprises HC having the amino acid sequence of SEQ NO. 9 and LC having the amino acid sequence of SEQ NO. 8, wherein glutamine (Q) at position 1 of SEQ NO. 9 is modified to pE, lysine (K) at position 450 of SEQ NO. 9 is clipped (e.g., via proteolytic degradation), and asparagine (N) at position 300 of SEQ NO. 9 is glycosylated, and the antibody comprises the following disulfide crosslinks: LC:C23-LC:C88; LC:C134-LC:C194; LC:C214-HC:C223; HC:C22-HC:C97; HC:C147-HC:C203; HC1:229-HC2:229 and HC1:232-HC2:232; HC:C264-HC:C324; and HC:C370-HC:C428. The numbering of the aforementioned cysteine ​​residues corresponds to their positions for Sequence Nos. 9 and 8, respectively. Additionally, the antibody comprises a glycan, wherein the glycan is a Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2 type glycan. In a preferred embodiment, the glycan is primarily a G0F and G1F type glycan. In some embodiments, the HC presented in Sequence No. 9 further comprises one or more of the following modifications:

[0054] The asparagine (N) at position 58 is deamidated;

[0055] Methionine (M) at position 71 is oxidized;

[0056] Methionine (M) at position 115 is oxidized;

[0057] Methionine (M) at position 225 is oxidized;

[0058] Aspartate (D) at position 283 is isomerized;

[0059] The asparagine (N) at position 318 is deamidated or contains succinimide;

[0060] Methionine (M) at position 361 is oxidized;

[0061] The asparagine (N) at position 387 is deamidated or contains succinimide;

[0062] Methionine (M) at position 431 is oxidized;

[0063] Glycine (G) at position 449 is absent;

[0064] Proline (P) at position 448 is amidated after the loss of C-terminal lysine and glycine.

[0065] Furthermore, as mentioned above, each heavy chain of antibody NI006 / ALXN2220 may contain a single N-linked glycosylation site in Asn300. The N-linked glycosylation structure is a fucosylated complex non-anteneric glycan having mainly zero galactose residues (G0F) (approx. 49%) or one galactose residue (G1F) (approx. 25%). Detailed glycosylation profiles are shown in Examples 2 and 3. 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 insightful information regarding the safety and efficacy of biotherapeutics.

[0066] Accordingly, in a preferred embodiment, the antibody of the present disclosure is an IgG antibody, particularly of the IgG1m3 subtype, having an N-glycosylated heavy chain, preferably the N-linked glycosylated site is Asn300.

[0067] Further detailed analysis of the glycan structure revealed that more than 85% of the glycans are fucosylated, specifically about 89% to 94% of the glycans. Additionally, it was found that about 80% to 90% of the glycans are part of the major fucosylated glycan types, meaning that most of the glycan structures attached to the antibody contain fucose residues. The fucosylation profile depends on the cell line in which the antibody is produced, and thus the fucosylation profile is specific to the antibody of the present invention. Furthermore, such a fucosylation profile is advantageous because fucosylation can improve the structural stability of the glycan structure on the antibody, which contributes to a longer in vivo half-life, which can reduce the frequency of administration and improve patient compliance. Moreover, highly fucosylated antibodies are typically less likely to induce an immune response in patients, as fucosylated glycans are common in human antibodies and are unlikely to be recognized as foreign substances. High levels of fucosylation are easier to consistently achieve using specific cell lines (such as CHO cells), are generally more reliable in large-scale manufacturing, and yield consistent products, which is critical for regulatory approval and therapeutic efficacy.

[0068] Accordingly, the antibody of the present disclosure preferably comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, preferably the major glycan types are G0F and G1F. More preferably, the antibody of the present disclosure comprises glycans, wherein more than 85% of the glycans are fucosylated, preferably about 85% to 95% of the glycans are fucosylated, and more preferably about 89% to 94% of the glycans are fucosylated.

[0069] Furthermore, it has been found that about 27% to 38% of the glycan, preferably about 27% to 38%, is galactosylated. Thus, the antibody exhibits a controlled glycosylation profile, which is important for quality control in antibody production because a consistent glycosylation pattern contributes to predictable efficacy, safety, and stability. Furthermore, galactosylation contributes to structural stability, which is important for maintaining a consistent therapeutic effect over time, which is advantageous for long-term antibody administration. Accordingly, in one embodiment, the antibody of the present disclosure comprises a glycan, wherein about 20% to 40%, preferably about 27% to 38%, of the glycan is galactosylated.

[0070] Analysis of the glycan structure revealed that approximately 1% to 4% of the glycans were mannose-containing glycans of the Man5 and Man31F types. Typically, high levels of mannose structures, such as Man5, can indicate incomplete processing in the glycosylation pathway; therefore, a low percentage (as in this case) is generally advantageous for therapeutic antibodies because it demonstrates that the antibody production process is well controlled and most glycans are processed into more complex structures. Furthermore, since mannose-rich glycans are less common in human antibodies and can sometimes increase immunogenicity because the immune system may recognize them as foreign, a low percentage of mannose is advantageous for the therapeutic application of antibodies.

[0071] Accordingly, in one embodiment, the antibody of the present disclosure comprises a glycan, wherein about 1% to 4% of the glycan is a mannose-containing glycan, preferably the glycan is of the Man5 and Man31F types, and more preferably about 1% to 4% of the glycan is of the Man5 type. In most of the analyzed batches, the mannose-containing glycan is present in much lower amounts, particularly 1% to 2%. Accordingly, more preferably, the antibody of the present disclosure comprises a glycan, wherein about 1% to 2% of the glycan is a mannose-containing glycan, preferably the glycan is of the Man5 and Man31F types, and more preferably about 1% to 2% of the glycan is of the Man5 type.

[0072] Finally, glycan analysis results revealed that about 0.5% to 2% of the glycan is sialylated, and thus, in one embodiment, the antibody of the present disclosure comprises glycan, wherein less than 2% of the glycan is sialylated, and preferably about 0.5% to 2% of the glycan is sialylated.

[0073] Regarding mannose-containing glycans, it was surprisingly found that reducing the pH dead band from 0.20 to 0.05 (pH was set to 6.9) led to a reduction in Man5 (see Example 3), which is advantageous as described above. In particular, the amount of Man5 type glycan is reduced by almost half. Accordingly, in a specific preferred embodiment, less than 3% of the glycan, preferably less than 2.9%, preferably less than 2.8%, preferably less than 2.7%, preferably less than 2.6%, preferably less than 2.5%, preferably less than 2.4%, preferably less than 2.3%, preferably less than 2.2%, preferably less than 2.1%, preferably less than 2.0%, preferably less than 1.9%, preferably less than 1.8%, preferably less than 1.7%, preferably less than 1.6%, preferably less than 1.5%, preferably less than 1.4%, preferably less than 1.3%, preferably less than 1.2%, preferably less than 1.1%, preferably less than 1.0%, preferably less than 0.9%, preferably less than 0.8%, preferably less than 0.7%, preferably less than 0.6%, preferably less than 0.5%, preferably less than 0.4%, preferably less than 0.3%, preferably less than 0.2%, preferably Less than 0.1%, preferably less than 0% is of the Man5 type. In a further preferred embodiment, about 2% of the glycan, preferably 2.5% to 1%, more preferably about 2% to 1% is of the Man5 type (see Table 9).

[0074] Accordingly, embodiments of the present disclosure further relate to methods and workflows for obtaining ALXN2220 having a desired product profile. Specifically, as described in detail in Example 3, reducing the pH dead band from 0.20 to 0.05 led to a significant reduction in acidic species (e.g., from about 37% to about 29% in ALXN2220 process A2) and resulted in a generally altered charged variant profile. Very surprisingly, reducing the pH dead band from 0.20 to 0.05 led to a significant reduction in Man5 levels, e.g., a reduction of about 50% (from about 3.8% to about 1.9%). Thus, embodiments of the methods and workflows described herein provide robust control of gomannose species in antibody drug substance batches without adverse effects associated with other product quality attributes, such as: major antibody species vs. HMW vs. Relative level (%) of LMW antibody species (as determined by SEC); level (%) of major antibody species as determined using capillary electrophoresis sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and level (%) of antibody heavy chains and light chains as determined using capillary electrophoresis sodium dodecyl sulfate (CE-SDS-R) under reducing conditions.

[0075] Further analysis revealed that the antibody samples included basic antibody variants and acidic antibody variants based on post-translational modifications. For example, antibodies containing deamidated asparagine residues at positions N58, N328, and / or N387 of the HC, antibodies containing sialylated N-glycosylation, galactosylated N-glycosylation, and HC cleavage between N58 and T59, as well as antibodies containing lysine glycosylation evenly distributed across all lysines in the HC and LC of the antibody, belong to the acidic species as further noted above, and antibodies containing aspartate isomerization at D283 of the HC, loss of lysine at K450 of the HC, and proline amidation (P448) with loss of glycine and lysine in the HC belong to the basic species as further noted above. Thus, in one embodiment, the antibody of the present disclosure is an acidic antibody variant. In another embodiment, the antibody of the present disclosure is a basic antibody variant. In another embodiment, the antibody of the present disclosure is a neutral antibody variant. In a preferred embodiment, the antibody of the present disclosure is an acidic antibody variant.

[0076] In some embodiments, the present disclosure relates to a recombinant antibody, for example, a suitable host cell, such as a bacterium (e.g., E. coli ( E. coli The present disclosure relates to antibodies produced through expression in )) cells, or cells of other microorganisms such as yeast cells; or mammalian cells, e.g., CHO cells. In embodiments of the present disclosure, the recombinant antibody comprises an N-linked glycosylated structure that is primarily a glycan having zero galactose residues (G0F) (about 49%) or a glycan having one galactose residue (G1F) (about 25%). Most preferably, the antibody comprises or is essentially composed of the glycosylated profile presented in Example 2 and mentioned above.

[0077] In an embodiment, the theoretical molecular weight (MW) of the antibody of the present disclosure is about 144.2 kDa, and the molecular weight determined by mass spectrometry (MS) is about 144.2 kDa (deglycosylated form). In an embodiment, the molecular weight of the antibody of the present disclosure as determined by mass spectrometry (MS) is 147.0 to 147.6 kDa (integrated IgG1).

[0078] Embodiments of the present disclosure further relate to a pharmaceutical composition comprising an antibody of the present disclosure as characterized above, having a molecular weight of about 150 kDa, preferably a MW of about 147 kDa.

[0079] In an embodiment, the experimentally determined pI of the antibody of the present disclosure is about 9.3 and the theoretical pI is about 8.4. In an embodiment, the experimentally determined quenching factor of the antibody of the present invention is 1.390 and the theoretically determined quenching factor is 1.438.

[0080] In some embodiments, the antibody of the present disclosure comprises a human IgG antibody comprising two identical heavy chains (HC) and two identical light chains (LC), wherein identity may be based on sequence identity, namely, the primary amino acid sequence of the heavy chain polypeptide sequence(s) constituting the antibody tetramer is, optionally or together with the primary amino acid sequence of the light chain polypeptide sequence(s) constituting the antibody tetramer. The four chains are stabilized by intra-chain and inter-chain disulfide bonds, wherein the positions of the disulfide crosslinks identified by Lys-C and trypsin digestion and subsequent LC-MS are as follows (see Example 2):

[0081] LC:C23-LC:C88;

[0082] LC:C134-LC:C194;

[0083] LC:C214-HC:C223;

[0084] HC:C22-HC:C97;

[0085] HC:C147-HC:C203;

[0086] HC1:229-HC2:229 and HC1:232-HC2:232;

[0087] HC:C264-HC:C324; and

[0088] HC:C370-HC:C428

[0089] (Here, the numbering of the cysteine ​​residues (C) corresponds to their positions in the antibody HC sequence of SEQ ID NO. 7 and the antibody LC sequence of SEQ ID NO. 8, wherein SEQ ID NO. 7 has N-terminal glutamine or pyroglutamic acid (X1).

[0090] Accordingly, in one embodiment, the antibody of the present disclosure preferably comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight disulfide crosslinks at the identified positions. In an embodiment, the antibody of the present disclosure comprises a plurality of, for example, all eight, disulfide (DS) crosslinks selected from crosslinks between light chains (LC) and LC; crosslinks between LC and heavy chains (HC); and crosslinks between HC and HC. In an embodiment, the antibody of the present disclosure comprises DS crosslinks selected from the following: (1) LC:C23-LC:C88; (2) LC:C134-LC:C194; (3) LC:C214-HC:C223; (4) HC:C22-HC:C97; (5) HC:C147-HC:C203; (6) HC1:229-HC2:229 and HC1:232-HC2:232; (7) HC:C264-HC:C324; and (8) HC:C370-HC:C428 (wherein LC specifies the antibody light chain; HC specifies the antibody heavy chain; C specifies the cysteine ​​amino acid; and the number specifies the position of said cysteine ​​in the antibody HC sequence of SEQ No. 7 containing glutamine or pyroglutamate in X1 (i.e., X1 is not absent in SEQ No. 7) and / or the antibody LC sequence of SEQ No. 8).

[0091] To avoid the formation of "anti-drug antibodies" (ADA) by a subject administered the antibody or its antigen-binding fragment described herein, the antibody is preferably a human or humanized antibody, typically human IgG, most preferably human IgG1. In a preferred embodiment, the antibody is a human IgG1m3 homologous type and preferably comprises a kappa LC constant region. Most preferably, the closest human genes / alleles of the variable domain are IGKV1-3901 (93.3%) + IGKJ101 (100%) and IGHV4-30-201 (89.5%) + IGHJ302 (100%).

[0092] Antibodies, like many other proteins, are secreted from cells via the co-translational translocation pathway. In eukaryotes, a signal peptide containing 5 to 30 amino acids located at the N-terminus of a nephrogenic protein is recognized by a signal recognition particle (SRP) in the cytosol while the protein is still being synthesized in the ribosome. Subsequently, the SRP delivers the SRP-ribosome-nephrogenic chain (SRP-RNC) complex to the SRP-receptor (SR) within the endoplasmic reticulum (ER) membrane. Then, a GTP-dependent mechanism delivers the RNC complex to a membrane-bound translocone, allowing the growing polypeptide chain to be translocated into the ER lumen. After crossing the ER membrane, the signal peptide is cleaved by a signal peptide peptidase (SPP), and the human IgG antibody consists of two identical heavy chains (HC) and two identical light chains (LC). Efficient expression of HC and LC requires signal peptides to transport HC and LC polypeptides into the ER for proper folding, assembly, and post-translational modification.

[0093] Accordingly, in one embodiment, the HC of the antibody of the present disclosure further comprises a signal peptide, wherein the signal peptide is derived from a human immunoglobulin heavy chain; and the LC of the antibody of the present disclosure further comprises a signal peptide, wherein the signal peptide is derived from a human immunoglobulin kappa light chain. Such signal peptides are generally known in the art, and their sequences may be derived from relevant databases, for example, the National Institutes of Health (NIH) database or the European Molecular Biology Laboratory (EMBL) database and the European Bioinformatics Institute (EBI) database of EMBL, respectively. Furthermore, WO 2014 / 058389 A1 discloses various signal peptides that, in principle, can be used for cloning. In this case, signal peptides as presented in SEQ ID NOs. 17 and 18 were used for the expression of the antibody of the present disclosure for the HC and LC, respectively, resulting in efficient expression of the HC and LC, which are then transported to the ER for proper folding, assembly, and post-translational modification, which leads to an antibody particularly suitable for formulation into a pharmaceutical composition.

[0094] Accordingly, in a preferred embodiment, the HC of the antibody of the present disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO. 17, wherein the sequence comprising the HC and the signal peptide is presented in SEQ ID NO. 19. Additionally or alternatively, the LC of the antibody of the present disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO. 18, wherein the sequence comprising the HC and the signal peptide is presented in SEQ ID NO. 20.

[0095] As is understood in the art, some antibodies may accept different signal peptides, while others are more restrictive. The signal peptides for the heavy and light chain antibody sequences represent, for example, the optimal signal peptide pair for the recombinant expression of NI006 / ALXN2220 in CHO cells.

[0096] The present disclosure further relates to a polynucleotide encoding the antibody of the present disclosure. In particular, the present disclosure relates to one or more polynucleotide(s), preferably two polynucleotides, encoding the antibody of the present disclosure, namely, the first polynucleotide comprises a nucleotide sequence encoding HC, and the second polynucleotide comprises a nucleotide sequence encoding LC of the antibody. Preferably, the present disclosure relates to a polynucleotide encoding antibodies HC and LC, wherein HC has the amino acid sequence of SEQ ID NO. 7 (unmodified, i.e., without glutamine or pyroglutamate (X1), with both lysine (X7) and glycine (X6) present, and X2, X3, X4, and X5 unmodified), and LC has the amino acid sequence of SEQ ID NO. 8. The present disclosure further relates to a polynucleotide encoding antibodies HC and LC, wherein HC has the amino acid sequence of SEQ ID NO. 9 (having a modification comprising deletion of C-terminal lysine and cyclization of N-terminal glutamine to pyroglutamate in the sequence of SEQ ID NO. 7), and LC has the amino acid sequence of SEQ ID NO. 8.

[0097] Accordingly, the present disclosure further relates to a polynucleotide encoding the aforementioned antibody, wherein the first polynucleotide comprises the nucleotide sequence presented in SEQ ID NO. 13 encoding antibody HC, and the second polynucleotide comprises the nucleotide sequence presented in SEQ ID NO. 14 encoding antibody LC.

[0098] The present disclosure further relates to codon-optimized nucleic acids for efficient expression, for example, in CHO cell lines. Accordingly, in one embodiment, the first polynucleotide of the present disclosure comprises a nucleotide sequence encoding antibody HC presented in SEQ ID NO. 15, and the second polynucleotide comprises a nucleotide sequence encoding antibody LC presented in SEQ ID NO. 16.

[0099] In one embodiment, the polynucleotide of the present disclosure further comprises a nucleotide sequence encoding a signal peptide. In particular, in one embodiment, the first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin kappa light chain. In a preferred embodiment, the nucleotide sequence of the first signal peptide is presented in SEQ ID NO. 21, wherein the first nucleotide sequence comprising the nucleotide sequence of said signal peptide has the sequence presented in SEQ ID NO. 23. Additionally, the nucleotide sequence of the second signal peptide is presented in SEQ ID NO. 22, wherein the first nucleotide sequence comprising the nucleotide sequence of said signal peptide has the sequence presented in SEQ ID NO. 24.

[0100] According to the present disclosure, the nucleic acid encoding a signal peptide for heavy chain and light chain antibody sequences represents an optimal signal peptide-encoding nucleic acid pair for recombinantly expressing the respective heavy chain and light chain polypeptide sequences of NI006 / ALXN2220 in, for example, CHO cells.

[0101] The present disclosure also relates to the following nucleic acid molecules:

[0102] (a) Polynucleotide(s) comprising the first nucleotide sequence presented in SEQ ID NO. 13 and the second nucleotide sequence presented in SEQ ID NO. 14;

[0103] (b) Messenger RNA (mRNA) equivalents of the first and second nucleotide sequences of (a);

[0104] (c) polynucleotide(s) comprising the first and second nucleotide sequences of (a) or the sequence complementary to the mRNA equivalent thereof of (b); or

[0105] (d) Polynucleotide(s) comprising the first and second nucleotide sequences of (a) or the degenerate sequence of the mRNA equivalent thereof of (b).

[0106] In a preferred embodiment, the polynucleotide(s) of (d) comprise the first nucleotide sequence presented in SEQ ID NO. 15 and the second nucleotide sequence presented in SEQ ID NO. 16. The degenerate sequences disclosed herein represent codon-optimized nucleic acid sequences that confer optimal recombinant expression of each heavy chain and light chain of the antibody in cells suitable for commercial production, e.g., CHO cells.

[0107] In one embodiment, the polynucleotide of the present disclosure further comprises a nucleotide sequence encoding a signal peptide. In particular, in one embodiment, the first nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a signal peptide derived from a human immunoglobulin kappa light chain. In a preferred embodiment, the nucleotide sequence of the first signal peptide is presented in SEQ ID NO. 21, wherein the first nucleotide sequence comprising the nucleotide sequence of said signal peptide has the sequence presented in SEQ ID NO. 23. Additionally, the nucleotide sequence of the second signal peptide is presented in SEQ ID NO. 22, wherein the first nucleotide sequence comprising the nucleotide sequence of said signal peptide has the sequence presented in SEQ ID NO. 24.

[0108] The present disclosure further relates to both a pre-treated form (e.g., pre-protein) and a treated (e.g., mature) form for each of the antibody heavy chain and light chain polypeptides encoded by the nucleic acid sequences presented in SEQ ID NO. 13 or 15 (encoding a pre-protein antibody heavy chain) and SEQ ID NO. 14 or 16 (encoding a pre-protein antibody light chain) mentioned above.

[0109] The present disclosure further relates to one or more expression vectors comprising the polynucleotide / nucleic acid molecule of the present disclosure and a host cell comprising said vector(s) and / or the polynucleotide / nucleic acid molecule of the present disclosure.

[0110] As described in Example 1, the antibody of the present disclosure is produced in CHO cells, particularly in CHO cell line K1. Thus, in one embodiment, the host cell is a non-human cell, preferably a CHO cell, most preferably a CHO-K1. For the recombinant production of the antibody, microorganisms, for example, E. coli cells or insect cells may also be used.

[0111] The present disclosure further relates to a method for producing an antibody of the present disclosure, the method comprising the steps of: culturing a host cell comprising a polynucleotide(s) of the present disclosure in the form of at least preferably a vector(s) of the present disclosure, preferably a CHO cell, most preferably a CHO K1 cell; and isolating an antibody from the culture medium.

[0112] As an example, in some embodiments, the method for preparing an antibody described herein produces an antibody comprising HC having the amino acid sequence for each of SEQ ID NOs 7 and 39, and LC having the amino acid sequence of SEQ ID NO. 8. In an embodiment, the antibody comprises HC having the amino acid sequence for each of SEQ ID NOs 7 and 39, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X I is asparagine or deamidated asparagine. In an embodiment, X II is methionine or oxidized methionine. In an embodiment, X IIIis methionine or oxidized methionine. In an embodiment, X IV is aspartate or iso-aspartate. In an embodiment, X V is asparagine or glycosylated asparagine. In an embodiment, X VI is methionine or oxidized methionine. In an embodiment, X VII is methionine or oxidized methionine. In additional embodiments, X1 is pE and / or X7 is absent.

[0113] The present disclosure further relates to a composition comprising the antibody of the present disclosure. Accordingly, in one embodiment, the composition comprises an antibody having or not having a PTM as defined above. In a preferred embodiment, the composition comprises a mixture of antibodies as defined above. As presented in Examples 2 and 3, about 99% to 100% of the antibodies present in a sample of a typical antibody composition have N-terminal pyroglutamic acid in the heavy chain, and about 96% of the antibodies have a loss of C-terminal lysine. Therefore, in one embodiment, about 99% (99% to 100%) of the antibodies in the formulation of the present disclosure have a heavy chain in which N-terminal pyroglutamic acid is modified from N-terminal glutamine, and / or about 96% (95% to 96%) of the antibodies do not have C-terminal lysine (or lack it). The absence of C-terminal lysine may be the result of proteolytic cleavage of the antibody heavy chain containing said lysine, or, for example, due to antibody manipulation through mutation or deletion of the triplet codon encoding the HC C-terminal lysine in the nucleic acid encoding said HC. Preferably, the antibody present in said composition further comprises N-glycosylated HC as defined above.

[0114] For example, in an embodiment, the composition comprises an antibody, wherein the antibody comprises a light chain having an amino acid sequence for SEQ ID NO. 7 and 39, respectively, and an amino acid sequence for SEQ ID NO. 8. In an embodiment, the antibody comprises an HC having an amino acid sequence for SEQ ID NO. 7 and 39, respectively, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X1 is PE in 99% to 100% of the antibody of the composition, preferably X1 is PE in 99.9% of the antibody of the composition. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X I is asparagine or deamidated asparagine. In an embodiment, X II is methionine or oxidized methionine. In an embodiment, X III is methionine or oxidized methionine. In an embodiment, X IV is aspartate or iso-aspartate. In an embodiment, X V is asparagine or glycosylated asparagine. In an embodiment, X VI is methionine or oxidized methionine. In an embodiment, X VII It is methionine or oxidized methionine.

[0115] In an embodiment, the composition comprises an antibody, wherein the antibody comprises a light chain having an amino acid sequence for SEQ ID NO. 7 and 39, respectively, and an amino acid sequence for SEQ ID NO. 8. In an embodiment, the antibody comprises an HC having an amino acid sequence for SEQ ID NO. 7 and 39, respectively, wherein the N-terminal residue X1 is absent or present as glutamine or pE. In an embodiment, X2 is methionine or oxidized methionine. In an embodiment, X3 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X4 is asparagine, deamidated asparagine, or asparagine comprising succinimide. In an embodiment, X5 is proline or amidated proline. In an embodiment, X6 is absent or glycine. In an embodiment, X7 is absent or lysine. In an embodiment, X7 is absent in about 95% to about 96% of the antibody of the composition, and preferably, X7 is absent in 95.8% of the composition. In an embodiment, X I is asparagine or deamidated asparagine. In an embodiment, X II is methionine or oxidized methionine. In an embodiment, X III is methionine or oxidized methionine. In an embodiment, X IV is aspartate or iso-aspartate. In an embodiment, X V is asparagine or glycosylated asparagine. In an embodiment, X VI is methionine or oxidized methionine. In an embodiment, X VII It is methionine or oxidized methionine.

[0116] In an embodiment, a portion of the antibody of the composition is fragmented at the HC through clipping between the asparagine at position 58 and the threonine at position 59, as presented for SEQ ID NOs. 7 and 39, respectively. In some embodiments, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the antibody of the composition is fragmented.

[0117] In addition, to a negligible degree and preferably in a negligible amount, some antibody species may be found in compositions that have undergone other post-translational modifications (PTMs), such as partial cleavage, oxidation, deamidation, succinimide or pyroglutamate formation, and isomerization. The PTMs identified as present in NI006 / ALXN2220 are referred to in Example 2. In particular, following the C-terminal lysine clipping and N-terminal cyclization mentioned above, the antibody may exhibit, for example, oxidation of methionine (M) at position HC 255; for example, deamidation of asparagine (N) at positions HC 318 and / or HC 387; for example, formation of asparagine (N) succinimide at position HC 318; and / or amidation of C-terminal proline (P) following the loss of C-terminal lysine and glycine.

[0118] In other words, the composition of the present disclosure comprises, in one embodiment, one or more antibodies comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein in one or more of the antibodies, the HC is preferably modified as follows:

[0119] Glutamine (Q) at position 1 is converted to pyroglutamate (pE);

[0120] The asparagine (N) at position 300 is glycosylated; and / or

[0121] Lysine (K) at position 450 is absent, preferably glutamine (Q) at position 1 is modified to pyroglutamate (pE), asparagine (N) at position 300 is glycosylated, and lysine (K) at position 450 is deleted.

[0122] In one embodiment, the composition of the present disclosure comprises one or more antibodies having the following additional modifications within the HC:

[0123] Methionine (M) at position 255 is oxidized;

[0124] The asparagine (N) at position 318 is deamidated or contains succinimide;

[0125] The asparagine (N) at position 387 is deamidated;

[0126] Proline (P) at position 448 is amidated;

[0127] Glycine (G) at position 449 is absent.

[0128] In an embodiment, the composition of the present disclosure comprises one or more antibodies having the following additional modifications within the HC:

[0129] The asparagine (N) at position 58 is deamidated;

[0130] Methionine (M) at position 71 is oxidized;

[0131] Methionine (M) at position 115 is oxidized;

[0132] Methionine (M) at position 255 is oxidized;

[0133] The aspartate (D) at position 283 is iso-aspartate;

[0134] Methionine (M) at position 361 is oxidized; and / or

[0135] Methionine (M) at position 431 is oxidized.

[0136] In a preferred embodiment, the antibody included in the composition of the present disclosure is produced through recombinant expression in CHO-K1 cells.

[0137] In a specific embodiment, the composition of the present disclosure comprises one or more antibodies comprising pyroglutamate instead of glutamine at the N-terminus of the heavy chain sequence, preferably in greater than 90%, preferably in about 99% to 100% of the antibody, glutamine (Q) at position 1 of SEQ No. 9 is modified to pyroglutamate. In a further specific embodiment, the composition of the present disclosure comprises one or more antibodies in which lysine (K) at position 450 of SEQ No. 9 is absent, preferably in greater than 90%, preferably in about 95% to 100%, more preferably in about 95% to 96% of the antibody, lysine (K) at position 450 of SEQ No. 9 is absent.

[0138] In additional specific embodiments, the composition of the present disclosure comprises one or more antibodies in which lysine (K) at position 450 of SEQ ID NO. 9 is absent, preferably in greater than 90% of the antibody, preferably in about 95% to 100%, more preferably in about 95% to 96%, lysine (K) at position 450 of SEQ ID NO. 9 is absent, and comprises one or more antibodies comprising pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, preferably in greater than 90% of the antibody, preferably in about 99% to 100%, glutamine (Q) at position 1 of SEQ ID NO. 9 is modified to pyroglutamate.

[0139] In a preferred embodiment, the asparagine residues (N) at positions 58, 318, and 387 in one or more antibodies are not deamidated and do not contain succinimide, and the composition does not contain an antibody in which the asparagine residues (N) at positions 58, 318, and / or 387 are each deamidated, or, for example, contains only a low amount of antibody of less than about 5% of the antibody. In a further preferred embodiment, the methionine residues at positions 71, 115, 255, 361, and 431 in one or more antibodies are not oxidized, and the composition does not contain an antibody in which the methionine residues at positions 71, 115, 255, 361, and / or 431 are oxidized, or, for example, contains only a low amount of antibody of less than about 5% of the antibody. In a further preferred embodiment, the aspartate at position 283 in one or more antibodies is not isomerized, and the composition does not contain antibodies in which the aspartate at position 283 is isomerized, or, for example, contains only a low amount of antibody of less than about 5%. In a further preferred embodiment, the glycine at position 449 in one or more antibodies is not absent, i.e., glycine is present, and the composition does not contain antibodies in which the glycine at position 449 is absent, or, for example, contains only a low amount of antibody of less than about 5%, i.e., the composition mainly contains antibodies in which glycine is present. Consequently, preferably, the proline at position 448 in one or more antibodies is not amidated, and the composition does not contain antibodies in which the proline at position 448 is amidated, or, for example, contains only a low amount of antibody of less than about 5%. More specifically, in an embodiment, the composition of the present disclosure comprises one or more antibodies in which the asparagine (N) at position 58 of SEQ ID NO. 9 is deamidated, preferably less than about 5%, preferably less than 2%, preferably less than about 1%, preferably about 0% to 1%.Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the asparagine (N) at position 318 of SEQ ID NO. 9 is deamidated, preferably in an amount of less than about 8%, preferably in an amount of about 6% to 9%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the asparagine (N) at position 387 of SEQ ID NO. 9 is deamidated, preferably in an amount of less than about 3%, preferably in an amount of about 1% to 3%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the methionine (M) at position 71 of SEQ ID NO. 9 is oxidized, preferably in an amount of less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably in an amount of about 0% to 1%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which methionine (M) at position 115 of SEQ ID NO. 9 is oxidized, preferably in less than about 5%, preferably in less than about 3%, preferably in less than about 2%, preferably in about 1% to 3%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which methionine (M) at position 255 of SEQ ID NO. 9 is oxidized, preferably in less than about 5%, preferably in less than about 3%, preferably in about 1% to 30%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which methionine (M) at position 361 of SEQ ID NO. 9 is oxidized, preferably in less than about 5%, preferably in less than about 2%, preferably in less than about 1%, preferably in about 0% to 1%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the methionine (M) at position 431 of SEQ ID NO. 9 is oxidized, preferably in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably in about 1% to 2%.Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the aspartate (D) at position 283 of SEQ ID NO. 9 is modified to iso-aspartate. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the proline (P) at position 448 of SEQ ID NO. 9 is amidated. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the glycine (G) at position 449 of SEQ ID NO. 9 is absent. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies that are fragmented through clipping between the asparagine at position 58 of SEQ ID NO. 9 and the threonine at position 59. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the heavy chain is glycosylated, preferably in an amount of less than about 6%, preferably less than about 5%, preferably in an amount of about 2% to 5%. Additionally or alternatively, the composition of the present disclosure comprises one or more antibodies in which the light chain is glycosylated, preferably in less than about 4%, preferably less than about 3%, preferably in about 1% to 3%.

[0140] Furthermore, the composition comprises one or more antibodies having a glycan profile defined in detail above.

[0141] As mentioned above, the composition of the present disclosure comprising an antibody having the indicated PTM is advantageous for therapeutic purposes because such composition can be prepared in a stable, safe, efficient, low immunogenic, functional, and reliably reproducible manner (see Example 4).

[0142] The same characterization can also be used to characterize the antibodies of the present disclosure.

[0143] The antibody of the present disclosure has been found to be particularly stable in an aqueous formulation of pH 5.8 comprising about 50 mg / mL or 100 mg / mL of the antibody, 20 mM histidine, sucrose at a concentration of 65 mg / mL (6.5% (w / v)) or 80 mg / mL (8% (w / v)), and polysorbate 80 (PS) at a concentration of 0.03% (w / v) (0.3 mg / mL). As shown in Example 4, the shelf life of a drug product formulated in the above-mentioned aqueous formulation is currently set to 24 months when stored at 5 ± 3°C under light protection.

[0144] Accordingly, the present disclosure relates to a composition comprising about 25 mg / mL to about 150 mg / mL of the antibody of the present disclosure, preferably about 50 mg / mL or 100 mg / mL of the antibody of the present disclosure, and further comprising about 20 mM concentration of histidine (e.g., L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), about 50 mg / mL to about 80 mg / mL, preferably 65 mg / mL (6.5% (w / v)) or 80 mg / mL (8% (w / v)) concentration of sucrose, about 0.01% (w / v) to about 0.5% (w / v), preferably about 0.03% (w / v) (0.3 mg / mL) concentration of polysorbate 80 (PS), and having a pH of about 5.3 to 6.3, preferably about 5.8. In a preferred embodiment, the antibody comprises two HCs having SEQ NO. 7 or 9 and two LCs having SEQ NO. 8, wherein preferably, in each HC, N-terminal glutamine (X1 of SEQ NO. 7 and Q at position 1 of SEQ NO. 9 for each) is modified to pyroglutamic acid, C-terminal lysine (X7 of SEQ NO. 7 and K at position 450 of SEQ NO. 9) is absent, the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is present at position Asn300 of SEQ NO. 9, wherein the amino acids at positions X2, X3, X4, and X5 of SEQ NO. 7 are modified or unmodified, preferably unmodified, or wherein methionine at position M255 of SEQ NO. 9 is not oxidized, and asparagine at positions N318 and N387 of SEQ NO. 9 is not deamidated and does not contain succinimide The proline at position P448 of sequence number 9 is not amidated, where glycine (X6 of sequence number 7 and G at position 449 of sequence number 9) is present and not clipped.In a preferred embodiment, the antibody comprises two HCs having SEQ ID NO. 39 or 9 and two LCs having SEQ ID NO. 8, wherein preferably, in each HC, N-terminal glutamine (X1 of SEQ ID NO. 39 and Q at position 1 of SEQ ID NO. 9 for each) is modified to pyroglutamic acid, C-terminal lysine (X7 of SEQ ID NO. 39 and K at position 450 of SEQ ID NO. 9) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is at position Asn300 (X of SEQ ID NO. 39). V and present at position 300 N of SEQ No. 9), wherein the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified, or wherein methionine at position M255 of SEQ No. 9 is not oxidized, asparagine at positions N318 and N387 of SEQ No. 9 is not deamidated and does not contain succinimide, proline at position P448 of SEQ No. 9 is not amidated, wherein glycine (X6 of SEQ No. 39 and G at position 449 of SEQ No. 9) is present and is not clipped, and wherein X at position 39 I , X II , X III , X IV , X VI , X VIThe amino acids are modified or unmodified, preferably unmodified, or wherein the methionine at positions M71, M115, M361, and M431 of SEQ ID NO. 9 is not oxidized, the asparagine at position N58 of SEQ ID NO. 9 is not deamidated, and the aspartate at position D283 of SEQ ID NO. 9 is not isomerized. In an alternative embodiment, the antibody consists of two heavy chains having SEQ ID NO. 9 and two light chains having SEQ ID NO. 8, wherein, preferably, the N-terminal glutamine in the heavy chain is modified to pyro-glutamic acid, the C-terminal lysine is removed or absent, the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is present at position Asn300.

[0145] In a preferred embodiment, the composition comprises one or more antibodies as defined above, that is, the composition comprises one or more antibodies in which lysine (K) at position 450 of SEQ ID NO. 9 is absent, preferably in more than 90% of the antibody, preferably about 95% to 100%, more preferably about 95% to 96%, lysine (K) at position 450 of SEQ ID NO. 9 is absent, and the composition comprises one or more antibodies comprising pyroglutamic acid instead of glutamine at the N-terminus of the heavy chain sequence, preferably in more than 90% of the antibody, preferably about 99% to 100%, glutamine (Q) at position 1 of SEQ ID NO. 9 is modified to pyroglutamate.

[0146] In a preferred embodiment, the asparagine residues (N) at positions 58, 318, and 387 in one or more antibodies are not deamidated and do not contain succinimide, and the composition does not contain an antibody in which the asparagine residues (N) at positions 58, 318, and / or 387 are each deamidated, or, for example, contains only a low amount of antibody of less than about 10%, preferably less than 5% of the antibody. In a further preferred embodiment, the methionine residues at positions 71, 115, 255, 361, and 431 in one or more antibodies are not oxidized, and the composition does not contain an antibody in which the methionine residues at positions 71, 115, 255, 361, and / or 431 are oxidized, or, for example, contains only a low amount of antibody of less than about 5% of the antibody. In a further preferred embodiment, the aspartate at position 283 in one or more antibodies is not isomerized, and the composition does not contain antibodies in which the aspartate at position 283 is isomerized, or, for example, contains only a low amount of antibody of less than about 5%. In a further preferred embodiment, the glycine at position 449 in one or more antibodies is not absent, i.e., glycine is present, and the composition does not contain antibodies in which the glycine at position 449 is absent, or, for example, contains only a low amount of antibody of less than about 5%, i.e., the composition mainly contains antibodies in which glycine is present. Consequently, preferably, the proline at position 448 in one or more antibodies is not amidated, and the composition does not contain antibodies in which the proline at position 448 is amidated, or, for example, contains only a low amount of antibody of less than about 5%.

[0147] In a preferred embodiment, the composition is an aqueous composition also referred to as a liquid formulation, and thus also includes water for injection.

[0148] As exemplified in Example 4, several stability tests performed with the formulation / pharmaceutical composition of the present disclosure demonstrate that the antibody remains stable under various conditions, e.g., at 40 ± 2°C and 75 ± 5% relative humidity (RH) for at least 1 month (stress stability study); at 25 ± 2°C / 60 ± 5% RH for at least 6 months (accelerated stability study); and at 5 ± 3°C for at least 12 to 18 months (long-term stability study). Furthermore, a shelf life of 24 months at 2 to 8°C when protected from light was determined for the antibody formulation.

[0149] Accordingly, in one embodiment, the composition may be characterized in that the antibody remains stable for at least one week, preferably for at least up to one month, at 40 ± 2°C and 75 ± 5% RH; at least one month, preferably for at least up to six months, at 25 ± 2°C / 60 ± 5% RH; and / or at least one month, preferably for at least up to 12 months, more preferably for at least up to 18 months, at 5 ± 3°C. In one embodiment, the pharmaceutical composition has a shelf life of 24 months at 2°C to 8°C when protected from light.

[0150] The stability of the formulation may be at least partially attributed to the PTM present in the constant region of the antibody, more specifically, in the heavy chain of the antibody of the present disclosure as defined above.

[0151] According to the present disclosure, a pharmaceutical formulation essentially characterized by a pH of 5.3 to 6.3, preferably 5.8 ± 0.1, comprising histidine buffer and, as excipients, sucrose, polysorbate, preferably polysorbate 80, and water for infusion / injection, was developed for a recombinant human monoclonal antibody specific to the disease-associated amyloidoid form of TTR, characterized by ALXN2220, also known as NI006, at a concentration of about 25 mg / ml to about 150 mg / ml, typically about 50 mg / ml.

[0152] As mentioned, the antibody formulation present at 50 mg / ml in 20 mM histidine, 6.5% or 8% (w / v) sucrose, and 0.03% (w / v) PS80 (pH 5.8) was identified in the examples as a leading candidate for further development; indeed, the antibody formulation present at 50 mg / ml in 20 mM histidine, 8% (w / v) sucrose, and 0.03% (w / v) PS80 (pH 5.8) was successfully used in clinical trials and thus represents an equivalent formulation that maintains physicochemical properties important for the stability and function of the antibody as tested and verified in Attached Example 4 as well as the most preferred embodiment of this disclosure. To vary the concentrations of components and / or alternative excipients in the preferred formulation, those skilled in the art should consider the following factors to ensure the stability and efficacy of the antibody:

[0153] Histidine: Acts as a buffer. Buffering capacity is related to histidine's ability to maintain pH near its pKa value. Since the pKa of histidine is approximately 6.0 at pH 5.8, it functions as a buffer, although slightly less efficient. Buffering capacity is also influenced by the concentration of the buffer and the proximity of pH to its pKa.

[0154] Sucrose: Stabilizes proteins during freeze / thaw cycles and adds osmolality. Sucrose significantly contributes to the osmolality of the solution, which is important for maintaining structural IgG antibodies.

[0155] High concentrations of protein of interest: 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 acid and glutamic acid, the amino groups of lysine and arginine, the imidazole groups of histidine, the hydroxyl groups of tyrosine, the thiol groups of cysteine, and terminal amino groups and carboxyl groups.

[0156] The composition of the present disclosure comprising the formulations mentioned above satisfies at least the approval criteria as set forth in Tables 25 and 26 (which may also be used to determine whether a composition / formulation with altered component concentrations and / or excipients replaced remains stable over a long period), and preferably, the composition of the present invention has a content of acidic species of antibody ≤ 40.0% and a content of basic species of antibody ≤ 15.0% and / or, as determined by iCIEF, after 6 months of storage at 5 ± 3°C, and a content of high molecular weight species ≤ 5.0% after 6 months of storage at 5 ± 3°C, as determined by SEC-HPLC.

[0157] It was found that during the manufacturing process of ALXN2220, as described in detail in Example 3, reducing the pH dead band from 0.20 to 0.05 significantly reduces acidic species, for example, from about 37% to about 29%. Accordingly, in one embodiment, the amount of acidic species in the composition of the present disclosure is about 30%, preferably 25% to 33%, more preferably 25% to 32%. It was further found that the charged variant profile is generally varied. Accordingly, in a further preferred embodiment, the amount of acidic species in the composition of the present disclosure is about 32% or less, preferably the amount of basic species is about 5% or less, and the amount of neutral species is about 63% or more.

[0158] Embodiments of the present disclosure relate to a method for producing an antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, comprising: (a) culturing a host cell, e.g., a Chinese hamster ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising polynucleotides encoding antibodies HC and LC, in a culture medium under conditions sufficient to express the antibody; (b) setting a pH set point and a pH dead band of the culture medium to adjust the levels of acidic, basic, and / or neutral antibody species in the culture medium; and (c) isolating antibodies HC and LC from the cell culture medium; and (d) optionally formulating the isolated antibody into a pharmaceutical formulation comprising the isolated antibody and a pharmaceutically acceptable carrier.

[0159] In a specific embodiment, the present disclosure relates to a method for producing an antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, comprising: (a) culturing a host cell, e.g., a Chinese hamster ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising polynucleotides encoding antibodies HC and LC, in a culture medium under conditions sufficient to express the antibody; (b) setting a pH set point and a pH dead band of the culture medium to adjust the levels of acidic, basic, and / or neutral antibody species in the culture medium; and (c) isolating antibodies HC and LC from the cell culture medium. and (d) optionally, the isolated antibody is formulated into a pharmaceutical formulation comprising the isolated antibody and a pharmaceutically acceptable carrier, wherein step (b) includes setting the pH set point to about 6.90 and setting the pH dead band to 0.05 to 0.10 to reduce the level of acidic antibody species; preferably, the pH dead band is set to about 0.05.

[0160] In some embodiments, the implementation of step (b), which includes the step of setting pH and pH dead band, reduces the level of acidic species in the cultured antibody preparation while also reducing the mannose 5 content.

[0161] In some embodiments, the implementation of step (b), comprising the step of setting the pH to 6.90 and the pH dead band to 0.05 to 0.10, preferably the step of setting the pH to 6.90 and the pH dead band to about 0.05, reduces the level of N-linked mannose-5 glycan (Man5) in the antibody preparation compared to a production method in which step (b) is implemented at a pH dead band of 6.90 and 0.20. In such embodiments, the reduction in the Man5 level is greater than >20%; more preferably, the reduction in the Man5 level is about 40% to 50% compared to the level of Man5 in the antibody preparation produced using the same production method, except that step (b) is implemented at a pH dead band of 6.90 and 0.20.

[0162] In some embodiments, the implementation of step (b), which includes the step of setting the pH to 6.90 and the pH dead band to 0.05 to 0.10, preferably the step of setting the pH to 6.90 and the pH dead band to about 0.05, is implemented in the generation step, which includes days 5 to 14 of the culture process, for example, days 5 to 14 after seeding the CHO-K1 cells.

[0163] In some embodiments, the implementation of step (b), comprising the step of setting the pH to 6.90 and the pH dead band to 0.05 to 0.10, preferably setting the pH to 6.90 and the pH dead band to about 0.05, controls the high mannose species in the antibody formulation without causing incidental adverse effects on the secondary quality attributes of the antibody formulation, for example, wherein the secondary quality attributes are selected from the following: (1) the relative level (%) of major antibody species vs. high molecular weight (HMW) vs. low molecular weight (LMW) antibody species in the formulation, for example, as determined by size exclusion chromatography (SEC); (2) the relative level (%) of major antibody species in the formulation, for example, as determined using capillary electrophoresis sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and / or (3) relative levels (%) of antibody heavy chains and light chains in the formulation, as determined, for example, using capillary electrophoresis sodium dodecyl sulfate (CE-SDS R) under reducing conditions.

[0164] Embodiments of the present disclosure relate to an antibody produced according to the above method, for example, an antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, produced by using a method comprising the following steps: (a) culturing a host cell, for example, a Chinese hamster ovary K1 (CHO-K1) cell, comprising one or more expression vectors or vector systems comprising polynucleotides encoding antibodies HC and LC, in a culture medium under conditions sufficient to express the antibody; (b) setting a pH set point and a pH dead band of the culture medium to adjust the levels of acidic, basic, and / or neutral antibody species in the culture medium—preferably, the adjustment comprises reducing acidic species in the antibody culture medium by setting the pH to 6.90 and the pH dead band to 0.05 to 0.10, preferably by setting the pH dead band to about 0.05—; and (c) isolating antibodies HC and LC from a cell culture medium; and (d) optionally, formulating the isolated antibodies into a pharmaceutical formulation comprising the isolated antibodies and a pharmaceutically acceptable carrier.

[0165] In an embodiment, the pH dead band is set to less than 0.2, preferably ≤ 0.19, preferably ≤ 0.18, preferably ≤ 0.175, preferably ≤ 0.17, preferably ≤ 0.16, preferably ≤ 0.15, preferably ≤ 0.14, preferably ≤ 0.13, preferably ≤ 0.125, preferably ≤ 0.12, preferably ≤ 0.11, preferably ≤ 0.1, preferably ≤ 0.09, preferably ≤ 0.08, preferably ≤ 0.075, preferably ≤ 0.07, preferably ≤ 0.06, most preferably ≤ 0.05. In a preferred embodiment, the pH dead band is set to about 0.1 to 0.05, preferably 0.05 or 0.1, most preferably 0.05.

[0166] In an embodiment, the antibody of the present disclosure is formulated into a high-concentration pharmaceutical composition for administration to a patient, for example, at pH 5.8. The pH is slightly lower than the pKa of histidine, which will still provide a buffering effect. For example, if the original buffer system has a concentration of 20 mM of histidine and a pH of 5.8, a person skilled in the art will be able to calculate the ratio of conjugate bases to acid using the Henderson-Hasselbalch equation. To maintain the pH, the concentrations of both the protonated form (HA) and the deprotonated form (A-) may be doubled up to 40 mM. Since changing the total concentration of the buffer will affect the ionic strength and osmolal concentration of the solution, which can affect the stability and solubility of the protein, the concentration and / or properties of an osmolality agent, in this case sucrose, may be adjusted, and the corresponding formulation may be tested according to the example.

[0167] As mentioned above, the heavy and light chain complementarity determining regions (VHCDR and VLCDR), including the heavy and light chain antigen-binding domains of NI006 / ALXN2220, are WO 2015 / 092077 A1 (designated as antibody NI-301.37F1) and literature [Michalon et al [., Nat. Commun. 12 (2021), 3142] (designated as antibody NI301A) is described. As disclosed in International Publication WO 2015 / 092077 A1, NI006 (NI-301.37F1) is characterized in particular by binding to agglutinated human wild-type transthyretin (wtATTR), which is illustrated in FIGS. 2 through 4 and 7 and described in Examples 3 through 6, and further described in the last paragraph on page 46. Additionally, International Publication WO 2015 / 092077 A1 discloses that NI006 (NI-301.37F1) does not bind to monomers and dimers of human natural transthyretin (TTR), as presented in Example 5 and FIG. 4. This binding profile is advantageous because the antibody selectively binds to aggregated wtTTR, allowing for the consideration of the treatment of hereditary transthyretin amyloidosis (hATTR) (formerly known as familial amyloid polyneuropathy, FAP) accompanied by polyneuropathy caused by mutations in the gene encoding TTR, as well as the treatment of wild-type transthyretin amyloidosis (wtATTR), known as senile systemic amyloidosis (SSA). Furthermore, this antibody poses no risk of interfering with the native monomer assembly into physiological tetramers. Additionally, this antibody [removes] ATTR deposits from patient-derived myocardium by macrophages in vitro ( ex vivo Not only was it found to be removed from ), but in vivo ( in vivoIt has also been revealed that it is eliminated in a dose- and time-dependent manner, where the biological activity of ATTR elimination involves antibody-mediated activation of phagocytic immune cells, including macrophages; literature [Michalon et al See ., Nat. Commun. 12 (2021), 3142.

[0168] Accordingly, the present disclosure relates to a composition, preferably a pharmaceutical composition, comprising, in an aqueous formulation further comprising L-histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection, an antibody of the present disclosure as defined above at a concentration of about 50 mg / ml to about 100 mg / ml, preferably about 50 mg / ml or 100 mg / ml, preferably about 50 mg / ml, for use in a method of treating TTR amyloidosis (ATTR) in a subject. Preferably, the composition is a composition of the present disclosure for use in a method of treating TTR amyloidosis (ATTR) in a subject, namely, a composition of the present disclosure that further comprises L-histidine (L-histidine and L-histidine monohydrochloride), sucrose, polysorbate 80, and water for injection, and the antibody of the present disclosure at a concentration of about 50 mg / ml to about 100 mg / ml, preferably at a concentration of about 50 mg / ml or 100 mg / ml, preferably at a concentration of about 50 mg / ml.

[0169] In a preferred embodiment, the formulation for each of the composition of the present disclosure and the composition for use according to the present disclosure is pH 5.8.

[0170] Furthermore, preferably, the formulations for each of the composition of the present disclosure and the composition for use according to the present disclosure have an osmolal concentration of ≥ 240 mOsm / Kg.

[0171] Furthermore, as mentioned above, the formulation of the composition is stable, and thus, in one embodiment, the pharmaceutical composition for use according to the present invention has a shelf life of 24 months at 2 to 8°C under conditions protected from light, and preferably exhibits the stability criteria mentioned above.

[0172] In one embodiment, the composition of the present disclosure and the composition for use according to the present disclosure comprise a formulation, said formulation such that the total volume is about 2 ml,

[0173] 50 mg or 100 mg of antibody,

[0174] 2.12 mg of L-histidine,

[0175] 5.56 mg of L-histidine monohydrochloride,

[0176] 160 mg of sucrose,

[0177] 0.6 mg of PS80, and

[0178] It is essentially composed of the main water.

[0179] If you wish to provide formulations of different volumes, for example, 2.25 ml, 20 ml, or 22.5 ml, the corresponding amounts of antibody and excipient can be calculated.

[0180] As mentioned in Example 1, the antibody NI006 / ALXN2220 can be generated in Chinese hamster ovary (CHO) cells. CHO cells are the most widely used mammalian cells for generating recombinant monoclonal antibodies due to their ability to perform post-translational modifications (PTMs) on antibody molecules that also typically occur in the human body. Through genetic engineering by mutagenesis, various CHO daughter cells of improved quality have been established. Among these variants are CHO-K1, CHO-S, CHO-DXB11, and CHO-DG44. Accordingly, in one embodiment, the antibody used in the composition of the present disclosure and the antibody used in the composition for use according to the present disclosure are generated in CHO cells, preferably CHO-K1 cells, and purified from cell culture medium for further use.

[0181] Furthermore, the compatibility of the antibody formulation and the clinically active material was evaluated. In particular, compatibility was evaluated with polyvinyl chloride IV bags, IV lines, and filters, as well as with polyvinyl chloride syringes and corresponding non-polyvinyl chloride materials when glucose or saline was used as a diluent. Three concentrations of 1 mg / mL, 20 mg / mL, and 50 mg / mL were evaluated, and the data provided in Example 4 indicate that the formulation is generally compatible with the clinically active material evaluated. Notably, in the saline group, visible particles were observed, indicating that NI006 / ALXN2220 is less stable in saline, and the data show that no substantial changes were observed in appearance, protein concentration, sub-visible particles, SEC-HPLC, and ELISA binding assays when diluted in glucose solution. Therefore, NI006 / ALXN2220 at concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL was found to be stable for 24 hours at 2°C to 8°C, followed by 6 hours at 25°C (total 30 hours), and was found to be compatible with the clinically used substance evaluated.

[0182] The most common route for administering monoclonal antibodies in the treatment is intravenous (IV) infusion. This method is desirable because it allows the antibody to be delivered directly into the bloodstream, ensuring immediate distribution throughout the body and enabling accurate administration.

[0183] Intravenous administration is particularly important due to the large molecular size of monoclonal antibodies, which generally prevents them from being effectively absorbed through the gut or skin. This means that oral or transdermal delivery methods are not suitable for this type of drug. Additionally, IV administration bypasses first-pass metabolism in the liver, which can significantly alter the drug's efficacy and safety profile.

[0184] Accordingly, the composition of the present disclosure and the composition for use according to the present disclosure are each specifically developed for IV administration and are therefore preferably formulations suitable for intravenous administration.

[0185] In some embodiments, the composition of the present disclosure and the composition for use according to the present disclosure are each not reconstituted from a lyophilized anti-TTR antibody or an antigen-binding fragment thereof, or are not further lyophilized.

[0186] In each of the compositions of the present disclosure and the compositions for use according to the present disclosure, since sucrose is used as a tonicity modifier and additional stabilizer for the antibody in the formulation, there is no need for NaCl, particularly when the primary purpose of NaCl is to stabilize the protein—which can be achieved by sucrose without increasing the ionic strength of the solution. Accordingly, the pharmaceutical compositions of the present disclosure are preferably essentially free of sodium chloride.

[0187] Additionally or alternatively, the composition of the present disclosure and the composition for use according to the present disclosure each essentially do not contain poloxamer (or, for example, are completely absent).

[0188] A formulation for each of the compositions of the present disclosure and the composition for use according to the present disclosure is preferably provided in a vial. In a preferred embodiment, the formulation is provided in a 2 ml glass vial, preferably a type I clear glass vial (preferably having a 12.5% ​​overfill (2.25 ml)), but other pharmaceutical vessels having different filling volumes are also included in the present disclosure, for example, a 20 ml vial, for example, a 20 ml glass vial (preferably having a 12.5% ​​overfill (22.5 ml)), or other vessels / vials designed such that the amount of the vial / vessel and the amount of antibody provided are each added to a flat dose—optional adding about 12.5%, which is provided, for example, by the overfill.

[0189] In a preferred embodiment, the formulation is provided in a 2 mL glass vial having an aluminum flip-off seal over a rubber stopper, to which preferably a volume of 2.25 mL is added.

[0190] In a preferred embodiment, the formulation for each of the composition of the present disclosure and the composition for use according to the present disclosure is a preservative-free concentrate for an infusion solution provided as a sterile, colorless to slightly yellowish, transparent to slightly milky liquid that is essentially free of visible particles, wherein, preferably, the concentrate for the solution is diluted in sterile glucose before administration as an intravenous infusion solution.

[0191] The present disclosure further relates to a corresponding pharmaceutical container comprising the composition / formulation of the present disclosure.

[0192] The present disclosure further relates to a stable aqueous formulation for use in a method for treating TTR amyloid cardiomyopathy (ATTR-CM), said formulation being

[0193] (i)

[0194] (a) Antibody of the present disclosure - preferably,

[0195] The antibody is composed of two HCs having SEQ NO. 7 or 9 and two LCs having SEQ NO. 8, wherein preferably, in each HC, N-terminal glutamine (X1 of SEQ NO. 7 and Q at position 1 of SEQ NO. 9 for each) is modified to pyroglutamic acid, C-terminal lysine (X7 of SEQ NO. 7 and K at position 450 of SEQ NO. 9) is absent, the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is present at position Asn300 of SEQ NO. 9, wherein the amino acids at positions X2, X3, X4, and X5 of SEQ NO. 7 are modified or unmodified, preferably unmodified, or wherein methionine at position M255 of SEQ NO. 9 is not oxidized, asparagine at positions N318 and N387 of SEQ NO. 9 is not deamidated and does not contain succinimide, and SEQ NO The proline at position P448 of 9 is not amidated, wherein glycine (X6 of SEQ ID NO. 7 and G at position 449 of SEQ ID NO. 9) is preferably present and not clipped. In a preferred embodiment, the antibody consists of two HCs having SEQ ID NO. 39 or 9 and two LCs having SEQ ID NO. 8, wherein preferably in each HC, the N-terminal glutamine (X1 of SEQ ID NO. 39 and Q at position 1 of SEQ ID NO. 9 for each) is modified to pyroglutamic acid, C-terminal lysine (X7 of SEQ ID NO. 39 and K at position 450 of SEQ ID NO. 9) is absent, the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is at position Asn300 (X of SEQ ID NO. 39 Vand present at position 300 N of SEQ No. 9), wherein the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified, or, wherein methionine at position M255 of SEQ No. 9 is not oxidized, asparagine at positions N318 and N387 of SEQ No. 9 is not deamidated and does not contain succinimide, proline at position P448 of SEQ No. 9 is not amidated, and glycine (X6 of SEQ No. 39 and G at position 449 of SEQ No. 9) is preferably present and not clipped, and wherein X at position 39 of SEQ No. 39 I , X II , X III , X IV , X VI , X VI The amino acids are modified or unmodified, preferably unmodified, or wherein methionine at positions M71, M115, M361, and M431 of SEQ ID NO. 9 is not oxidized, asparagine at position N58 of SEQ ID NO. 9 is not deamidated, and aspartate at position D283 of SEQ ID NO. 9 is not isomerized; alternatively, the antibody consists of two heavy chains having SEQ ID NO. 9 and two light chains having SEQ ID NO. 8, wherein preferably, in the heavy chain, N-terminal glutamine is modified to pyroglutamic acid, C-terminal lysine is removed or absent, the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is present at position Asn300 -,

[0196] (b) histidine at a concentration of about 20 mM,

[0197] (c) Sucrose at a concentration of about 8% (w / v),

[0198] (d) PS80 at a concentration of about 0.03% w / v,

[0199] (e) essentially composed of water for injection,

[0200] (ii) The pH is about 5.8 and;

[0201] (iii) contained in a 2 mL disposable vial containing about 100 mg of antibody or a 20 mL disposable vial containing 1000 mg of antibody;

[0202] (iv) Before administration, dilute with a sterile glucose solution, preferably to an antibody concentration of 1 mg / mL or more;

[0203] (v) Administered by intravenous infusion.

[0204] The present disclosure further relates to a kit comprising one or more containers of the present disclosure and means for delivering the formulation / composition to a human subject, optionally the means comprising an infusion bag or a syringe. In a preferred embodiment, the formulation is a preservative-free, clear to milky white, colorless to pale yellow solution provided in a vial at 100 mg / 2 mL.

[0205] The present disclosure further relates to a manufactured article comprising one or more of the container(s) and a label of the present disclosure, wherein the label specifies that the antibody is indicated for the treatment of ATTR, particularly ATTR cardiomyopathy (ATTR-CM), e.g., wild-type or hereditary transthyretin-mediated amyloid cardiomyopathy (wtATTR-CM or hATTR-CM). In a preferred embodiment, the kit includes an accompanying document specifying that the antibody should be administered intravenously, which may accommodate, e.g., instructions for IV administration. In a preferred embodiment, the container(s) are glass vials as defined above.

[0206] In a further preferred embodiment, the kit may additionally comprise a diluent. In an embodiment, the diluent comprises a 5% glucose solution, wherein the glucose solution may be provided in an additional container. In a preferred embodiment, the antibody formulation is a preservative-free, clear to milky white, colorless to pale yellow solution provided in a vial at 100 mg / 2 mL.

[0207] The present disclosure further relates to a method for treating ATTR, preferably ATTR cardiomyopathy (ATTR-CM) and / or ATTR polyneuropathy (ATTR-PN), the method comprising the step of administering the antibody and composition of the present disclosure, respectively, to a subject requiring the method, preferably the administration is performed intravenously. Preferably, the antibody / composition is diluted and administered by intravenous infusion over approximately 1 to 2 hours, wherein the dilution is preferably performed in a 5% glucose solution.

[0208] In some embodiments, patients to be treated with the antibody of the present disclosure or a pharmaceutical composition containing it are pre-screened to confirm, for example, that they have wtATTR-CM or hATTR-CM, and treatment is selectively provided to such patients who are confirmed positive.

[0209] In some embodiments, the subject has been previously treated with or is currently being provided with a disease modifier, such as TTR tetramer stabilizer, e.g., tafamidis (VYNDAQEL® or VYNDAMAX®) or acoramidis (AG10). Currently, oral disease-modifying tafamidis is the only approved pharmacological treatment that specifically targets ATTR-CM in both wild-type and genetic forms.

[0210] The present disclosure further provides a method for manufacturing the antibody of the present disclosure and a corresponding drug product, respectively.

[0211] A method for generating an antibody or its antigen-binding fragment comprises the following steps:

[0212] a) cloning the nucleic acid molecule and polynucleotide of the present disclosure, comprising a first nucleotide sequence and preferably a nucleotide sequence of a first signal peptide, respectively, into an expression vector, and cloning the nucleic acid molecule and polynucleotide of the present disclosure, comprising a second nucleotide sequence and preferably a nucleotide sequence of a second signal peptide, respectively, into an expression vector—wherein the first and second nucleotide sequences may be provided in the same or different expression vectors—;

[0213] b) a step of transforming the expression vector(s) into a non-human host cell, preferably a CHO cell, more preferably a CHO-K1 cell;

[0214] c) a step of culturing host cells under conditions that enable the expression of immunoglobulin chains including heavy chains and light chains; and

[0215] d) a step of isolating the immunoglobulin chain and the generated IgG antibody from the culture medium, respectively, and optionally, generating a Fab fragment by using enzymatic degradation including papain cleavage to generate an antigen-binding fragment of the antibody by degrading the IgG antibody.

[0216] In an embodiment, the method of the present disclosure comprises the step of culturing host cells, particularly CHO-K1 cells, to a pH dead band of less than 0.2, preferably ≤ 0.19, preferably ≤ 0.18, preferably ≤ 0.175, preferably ≤ 0.17, preferably ≤ 0.16, preferably ≤ 0.15, preferably ≤ 0.14, preferably ≤ 0.13, preferably ≤ 0.125, preferably ≤ 0.12, preferably ≤ 0.11, preferably ≤ 0.1, preferably ≤ 0.09, preferably ≤ 0.08, preferably ≤ 0.075, preferably ≤ 0.07, preferably ≤ 0.06, most preferably ≤ 0.05. In a preferred embodiment, the pH dead band is set to about 0.1 to 0.05, preferably 0.05, or 0.1, most preferably 0.05. Preferably, the method of the present disclosure comprises the step of culturing host cells, particularly CHO-K1 cells, to a pH dead band of pH 6.9 to less than 0.2, preferably ≤ 0.19, preferably ≤ 0.18, preferably ≤ 0.175, preferably ≤ 0.17, preferably ≤ 0.16, preferably ≤ 0.15, preferably ≤ 0.14, preferably ≤ 0.13, preferably ≤ 0.125, preferably ≤ 0.12, preferably ≤ 0.11, preferably ≤ 0.1, preferably ≤ 0.09, preferably ≤ 0.08, preferably ≤ 0.075, preferably ≤ 0.07, preferably ≤ 0.06, most preferably ≤ 0.05. In a preferred embodiment, the pH dead band is set to about 0.1 to 0.05, preferably 0.05 or 0.1, most preferably 0.05. In an embodiment, the pH is set to pH 6.9 with the indicated pH dead band.

[0217] Additionally, the above method may be used to produce the pharmaceutical composition of the present disclosure and a corresponding drug product. In particular, after producing an antibody, the method further comprises the following steps:

[0218] e) a step of formulating the antibody into an aqueous solution comprising an antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v (wherein the formulation has a pH of about 5.8) to produce a pharmaceutical composition comprising the antibody; and optionally,

[0219] f) a step of filling the composition into a vial, and additionally optionally,

[0220] g) A step of packaging the pharmaceutical composition and the vial, respectively, in a kit, along with instructions for the administration of the antibody to human patients, e.g., intravenous administration.

[0221] As mentioned above, the antibody of the present invention has been found to remove ATTR deposits from patient-derived myocardium in vitro by macrophages, as well as to remove them in vivo in a dose- and time-dependent manner from mice transplanted with patient-derived ATTR fibrils, wherein the biological activity for ATTR removal comprises antibody-mediated activation of phagocytic immune cells including macrophages; literature [Michalon et al See ., Nat. Commun. 12 (2021), 3142.

[0222] To further investigate the mechanism of macrophage-mediated amyloid depletion, a high-resolution live cell imaging assay was developed. This assay involves the steps of incubating cardiac tissue sections containing ATTR with THP-1-derived macrophages in the presence of anti-TTR antibodies, followed by performing live cell imaging.

[0223] In one approach, the method comprises the steps of staining amyloid deposits within a tissue section with the amyloid-specific red fluorescent dye Amytracker 680 and labeling ALXN2220 with the green fluorescent dye Vivotag-680. After incubating the stained tissue section with macrophages in the presence of the labeled ALXN2220, high-resolution live cell imaging was performed as described in Example 5. Superposition of fluorescent images visualizing the red fluorescent dye used to stain the amyloid deposits or the green fluorescent dye used to label the antibody showed that the same part of the tissue was stained with the red fluorescent dye that directly stains the amyloid deposits, and with the green fluorescent dye that stains the amyloid deposits by binding the labeled antibody to the amyloid deposits; see Fig. 7. Thus, binding of the antibody to ATTR in the tissue section was confirmed.

[0224] In another approach, the method comprises the step of staining amyloid deposits within tissue sections with the amyloid-specific red fluorescent dye Amytracker 680. ALXN2220 was not labeled. After incubating the stained tissue sections with macrophages in the presence of antibodies, high-resolution live cell imaging was performed as described in Example 5. As shown in Fig. 8, ALXN2220 triggered amyloid phagocytosis by macrophages. Dotted and intracellular red fluorescence patterns indicated the presence of ATTR amyloid within phagocytic vesicles, thus demonstrating macrophage-mediated ATTR internalization.

[0225] In an additional approach, the method comprises the step of labeling ALXN2220 with the green fluorescent dye Vivotag-680. After incubating unstained tissue sections with macrophages in the presence of the labeled antibody, high-resolution live cell imaging was performed as described in Example 5, and the fluorescence signals shown in Figures 9 and 10 indicate amyloid staining due to antibody binding to ATTR. As shown in Figures 9 and 10, punctate fluorescence signals separated from the amyloid fluorescence signal over time, or thin and elongated fluorescence signals separated from adjacent cardiomyocytes, as well as punctate fluorescence signals, indicate macrophage-mediated amyloid fragmentation followed by the separation of ATTR deposits from adjacent cardiomyocytes and amyloid fragmentation.

[0226] Therefore, it was revealed for the first time that live cell imaging is a suitable method for detecting antibody binding to amyloid deposits and visualizing the removal of amyloid deposits through the activation of phagocytic macrophages. These findings open up the possibility of developing screening and validation methods for anti-amyloid antibodies and additional amyloid-depleting compounds using live cell imaging.

[0227] Accordingly, in one aspect, the present invention relates to a method for verifying an amyloid-depleting drug (also referred to as an amyloid-depleting compound), the method comprising the steps of incubating a tissue section containing amyloid deposits with macrophages in the presence of an amyloid-depleting drug—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye—and performing high-resolution live cell imaging, wherein (a) the superposition of the antibody fluorescent signal and the amyloid fluorescent signal indicates binding of the amyloid-depleting drug to the amyloid; (b) the presence of a dot-like intracellular fluorescent signal in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of the dot-like fluorescent signal from the amyloid fluorescent signal indicates macrophage-mediated amyloid fragmentation. Therefore, live cell imaging can be used to visualize the binding of an amyloid-depleting drug to amyloid deposits, macrophage-mediated amyloid internalization, and macrophage-mediated amyloid fragmentation, wherein these mechanisms led to intracellular amyloid degradation. Thus, if any one of (a) to (c), preferably all of (a) to (c), more preferably at least one of (b) and (c), or if (b) and (c) are observed during live cell imaging, it is verified that the amyloid-depleting drug actually possesses amyloid-depleting activity.

[0228] To confirm that the results are specific, that is, attributed to the activity of the amyloid-depleting drug, the observed mechanisms (binding of the amyloid-depleting drug to amyloid deposits, macrophage-mediated amyloid internalization, and macrophage-mediated amyloid fragmentation) should not be observed when tissue sections containing amyloid deposits and macrophages are incubated in the presence of the amyloid-depleting drug, for example, when they are incubated in the presence of a control compound. The control compound preferably belongs to the same class of substances as the amyloid-depleting drug, preferably the same class of substances as the antibody, but exhibits non-specific binding. The same applies to the methods further described below.

[0229] In another aspect, the present invention relates to a screening method for identifying and selectively obtaining an amyloid-depleting drug from a plurality of test compounds, the method comprising the steps of incubating a tissue section having amyloid deposits with macrophages in the presence of a test compound—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the test compound is labeled with a fluorescent dye—and performing high-resolution live cell imaging, wherein (a) the overlap of the test compound fluorescent signal and the amyloid fluorescent signal indicates binding of the amyloid-depleting drug to the amyloid; and (b) the presence of an intracellular fluorescent signal as a dot in phagocytic vesicles indicates macrophage-mediated amyloid internalization; (c) The sequential separation of point-like fluorescence signals from the amyloid fluorescence signal indicates macrophage-mediated amyloid fragmentation, wherein the presence of any one of items (a) to (c), preferably all of (a) to (c), or at least one of (b) and (c), preferably (b) and (c), indicates the suitability of the test compound as an amyloid-depleting drug.

[0230] The present invention further relates to a method for screening amyloid-depleting drugs for the ability to bind to amyloid, mediate macrophage recruitment to amyloid deposits, and then perform amyloid fragmentation and internalization, i.e., the ability to activate macrophage-mediated amyloid fragmentation and internalization, thereby leading to the intracellular degradation of amyloid, wherein the method comprises the steps of incubating a tissue section containing amyloid deposits with macrophages in the presence of an amyloid-depleting drug—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye—and performing high-resolution live cell imaging, wherein (a) the overlap of the antibody fluorescent signal and the amyloid fluorescent signal indicates binding of the amyloid-depleting drug to amyloid; and (b) the presence of an intracellular fluorescent signal as a dot in phagocytic vesicles indicates macrophage-mediated amyloid internalization; (c) The sequential separation of punctate fluorescence signals from the amyloid fluorescence signal indicates macrophage-mediated amyloid fragmentation.

[0231] In one embodiment, the method of the present invention comprises testing a control sample, for example, incubating a tissue section with macrophages in the presence of a control. To be recognized as an amyloid-depleting drug, the test substance used in the screening method must have enhanced binding of the amyloid-depleting drug to amyloid, enhanced amyloid internalization, and / or enhanced amyloid fragmentation compared to a control, and / or, to be suitable for the treatment of amyloidosis or amyloid-related diseases, the amyloid-depleting drug must have enhanced binding of the amyloid-depleting drug to amyloid, enhanced amyloid internalization, and / or enhanced amyloid fragmentation compared to a control. The control sample may be any control exhibiting non-specific binding to amyloid and / or macrophages.

[0232] The present invention also relates to a method for producing a pharmaceutical composition of an amyloid-depleting drug, the method comprising: (i) optionally producing an amyloid-depleting drug; (ii) applying the amyloid-depleting drug to a method as defined above; (iii) using the information obtained in step (ii) as part of an evaluation of whether the amyloid-depleting drug can be used as a pharmaceutical composition; and optionally, formulating the amyloid-depleting drug found to be useful as a pharmaceutical composition in step (iii) using a pharmaceutically acceptable carrier, such as a buffer, a tonicity agent and / or a surfactant, most preferably all three of these components.

[0233] In another aspect, the present invention relates to a method for characterizing, verifying, developing, and / or quality controlling, e.g., batch control of an amyloid-depleting drug, wherein the method comprises: (i) optionally generating, providing, an amyloid-depleting drug; (ii) applying the amyloid-depleting drug to a method as described above; (iii) conveying the information obtained in (i) to a customer, contracting party, or collaborating partner and / or selecting a drug determined to be a suitable amyloid-depleting drug; and optionally, (iv) using the amyloid-depleting drug, or a pharmaceutical composition comprising the amyloid-depleting drug, for the treatment of amyloidosis or amyloid-related diseases.

[0234] Combination therapies for the treatment of amyloidosis are also of interest. For example, as disclosed in WO 2021 / 228987 A1, the combination of an anti-TTR antibody as an amyloid-depleting drug with TTR tetramer stabilizers such as tafamidis and diflunisal, or TTR gene silencing agents, is a promising approach for the treatment of TTR amyloidosis. In the development of combination therapies, the effects of the drugs on each other must be carefully tested to avoid unwanted effects, such as reduced therapeutic efficacy.Accordingly, the present invention also relates to a method for analyzing the effect of an agent, e.g., a second drug, a tracer, radiation, or a label on the amyloid-depleting activity of an amyloid-depleting drug, preferably the agent being a second drug, particularly a second drug for treating amyloidosis or amyloid-related diseases, or an analgesic, e.g., a non-steroidal anti-inflammatory drug, such as ibuprofen or paracetamol, and the method comprising the steps of: incubating a tissue section having amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of an amyloid-depleting drug and an agent; and performing high-resolution live cell imaging, wherein (a) the superposition of the antibody fluorescence signal and the amyloid fluorescence signal indicates the binding of the amyloid-depleting drug to amyloid; and (b) the presence of an intracellular fluorescence signal as a dot in phagocytic vesicles indicates macrophage-mediated amyloid internalization; (c) A sequential separation of point-like fluorescence signals from the fluorescence signal of amyloid indicates macrophage-mediated amyloid fragmentation, wherein, preferably, in contrast to a control group in which a tissue section containing amyloid deposits is incubated with macrophages and an amyloid-depleting drug without an agent, a substantially unchanged fluorescence pattern of items (a) to (c) indicates the suitability of the combination of an amyloid-depleting drug and an agent in the treatment of amyloidosis or amyloid-related diseases, and a substantially changed fluorescence pattern of any one of items (a) to (c) indicates the effect of the agent on the amyloid-depleting activity of the amyloid-depleting drug.

[0235] Enhanced fluorescence signals emitted from amyloid deposits indicate that the binding of amyloid-depleting drugs is more efficient in the presence of an agent, thereby demonstrating a synergistic effect on the amyloid-depleting activity of the amyloid-depleting drugs. Reduced fluorescence signals emitted from amyloid deposits indicate that the binding of amyloid-depleting drugs is less efficient in the presence of an agent, thereby demonstrating a detrimental effect on the amyloid-depleting activity of the amyloid-depleting drugs.

[0236] Additionally or alternatively, enhanced intracellular fluorescence signaling in phagocytic vesicles indicates enhanced macrophage-mediated amyloid internalization and thus exhibits a synergistic effect on the amyloid-depleting activity of amyloid-depleting drugs. Reduced intracellular fluorescence signaling in phagocytic vesicles indicates reduced macrophage-mediated amyloid internalization and thus exhibits a detrimental effect on the amyloid-depleting activity of amyloid-depleting drugs.

[0237] Additionally or alternatively, enhanced separation of punctate fluorescence signals from the amyloid fluorescence signal indicates enhanced macrophage-mediated amyloid fragmentation and thus exhibits a synergistic effect on the amyloid depletion activity of amyloid-depleting drugs. Reduced separation of punctate fluorescence signals from the amyloid fluorescence signal indicates reduced macrophage-mediated amyloid fragmentation and thus exhibits a detrimental effect on the amyloid depletion activity of amyloid-depleting drugs.

[0238] In a preferred embodiment of the method of the present invention, the amyloid-depleting drug is an anti-amyloid antibody, most preferably an anti-TTR antibody, and thus the amyloid deposit is preferably a TTR amyloid deposit, and the amyloidosis and amyloid-related disease is preferably TTR amyloidosis or a TTR amyloid-related disease, most preferably cardiac TTR amyloidosis. Accordingly, the tissue section is preferably a cardiac tissue section. In this regard, the agonist is preferably a second drug, which is preferably a drug useful for the treatment of TTR amyloidosis, for example, a TTR tetramer stabilizer, such as tafamidis or diflunisal, or a TTR gene silencing agent.

[0239] The amyloid-depleting drug to be verified by the method of the present invention, characterized by the method of the present invention, subjected to quality control using the method of the present invention, or analyzed for the effect of an agent, e.g., a second drug, on amyloid-depleting activity is preferably the anti-TTR antibody of the present invention, most preferably the antibody ALXN2220 as characterized above. The antibody may also be used as a positive control in the method of the present invention, particularly in the screening and confirmation method. Thus, the method of the present invention involves the use of a positive control, which is preferably the antibody of the present invention as defined above, most preferably the antibody ALXN2220.

[0240] In a preferred embodiment, high-resolution live cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. When amyloid in a tissue section is stained with an amyloid-specific fluorescent dye and an antibody is also labeled with a fluorescent dye, preferably two different dyes are used, preferably dyes of different colors, for example, red and green fluorescent dyes are used.

[0241] Tissue sections containing amyloid are preferably derived from subjects suffering from amyloidosis or amyloid-related diseases, preferably from TTR amyloidosis, and most preferably from cardiac TTR amyloidosis.

[0242] The present invention further relates to a kit useful for carrying out the method of the present invention, said kit comprising at least a first fluorescent dye for amyloid staining and / or a second fluorescent dye for labeling an amyloid-depleting drug, and optionally comprising instructions for use or consumables useful for carrying out said method, e.g., microdishes. Preferably, the kit may also include negative and / or positive controls, wherein the positive control may be, for example, an antibody as defined above, preferably antibody ALXN2220.

[0243] In the past, additional assays were developed to determine the removal or reduction of amyloid fibrils when amyloid-depleting drugs, particularly anti-TTR antibodies, were administered to animals in non-human animal models of patient-derived amyloid xenografts (PDAX) containing amyloid fibril implants (see WO 2020 / 094883 A1), or to determine the potency of target antigen-binding molecules, particularly anti-TTR antibodies, in mediating the phagocytosis of amyloidogenic TTRs (see WO 2023 / 099788 A1). These assays can be combined with the method of the present invention to improve the accuracy of the results. For example, the method of the present invention may be performed in conjunction with a corresponding method using a patient-derived amyloid xenograft (PDAX) non-human animal model, wherein the animal is characterized by the implantation of amyloid fibrils derived from the tissue or organ of a patient suffering from amyloidosis or amyloid-related disease, wherein the amyloid and amyloid fibrils each contain amyloid transthyretin (ATTR), and the amyloid fibrils are implanted subcutaneously or subcapsulated, or implanted in the kidney, peritoneum, muscle, brain, ventricles, nerve, eye, tongue, or heart, and the corresponding method comprises the steps of administering an amyloid-depleting drug or test substance to the PDAX non-human animal model and determining amyloid fibrils in the model, wherein, upon administration of the drug or test substance, the accelerated removal or reduction of amyloid fibrils compared to a control group indicates the suitability of the amyloid-depleting drug and the amyloid-depleting activity of the test substance for the treatment of amyloidosis or amyloid-related disease, respectively.

[0244] definition

[0245] To avoid any doubt, expressions such as “in some embodiments,” “in specific embodiments,” “in specific cases,” “in some cases,” “in additional embodiments,” “in one embodiment,” “in additional aspects,” “in a first aspect,” “in a second aspect,” and similar expressions are used, and it is emphasized that any of the embodiments described herein should be read with the intent to combine each of the features of such embodiments, and that the disclosure should treat combinations of features of such embodiments and aspects in the same manner as those mentioned in one embodiment. This applies to any combination of embodiments and features shown in the appended claims and embodiments, which are also intended to be combined with the features of the corresponding embodiments disclosed in the description, where, for the sake of consistency and brevity only, each combination of embodiments and features that may actually be formed by (multiple) dependencies should be considered as explicitly disclosed and should not be considered as having selected one of different options. In this regard, those skilled in the art will understand that the embodiments and features disclosed in the Examples are intended to be generalized to any anti-TTR antibody and equivalent having substantially the same characteristics.

[0246] As used herein, the term “about” refers to a value within ± 10%, preferably ± 5%, of the cited value. For example, “about 8%” may mean any percentage from 7.2% to 8.8%, preferably any percentage from 7.6% to 8.4%. In another example, “about 2 mL” may mean any volume from 1.8 mL to 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). With respect to antibody amounts as mentioned in this specification, e.g., 50 mg / mL or 100 mg / mL, the term “about” refers to concentrations in the range of 45 mg / mL to 50 mg / mL, preferably 48 mg / mL to 52 mg / mL, and concentrations in the range of 90 mg / mL to 113 mg / mL, preferably 96 mg / mL to 113 mg / mL. Accordingly, even if the term “about” is not explicitly used, the concentration ranges indicated above 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, as these ranges are within experimental variation.

[0247] In connection with the present disclosure, the term “and / or” is understood to mean that all members of the group connected by the term “and / or” are disclosed in any combination, alternatively with respect to one another, and in each case cumulatively with respect to one another. This means that the expression “A, B and / or C” should be understood as the following disclosures: a) A or B or C; or b) (A and B); or c) (A and C); or d) (B and C); or e) (A, B, and C).

[0248] As used herein, “antibody” refers to a group of immunoglobulin molecules that specifically bind to a target antigen. The term “antibody” is understood to include not only single molecular entities but also variants of antibodies that may arise from post-translational modifications such as glycosylation, deamidation, lysine clipping, and pyroglutamate formation. These modifications may create heterogeneous populations of individual molecules within a batch that differ in the number or type of modifications present. Accordingly, the term “antibody” refers to this entire group, including such modified and unmodified forms.

[0249] With respect to the formulations / pharmaceutical compositions of this disclosure, the phrases "essentially free of NaCl" and "substantially free of NaCl" mean that the pharmaceutical composition / formulation contains no NaCl or contains only trace amounts of NaCl that are considered negligible for the intended use of the product, i.e., NaCl present at levels low enough not to affect the performance, stability, safety, or efficacy of the formulation. Furthermore, the phrases also refer to cases where NaCl is not intentionally added, but due to the presence of other excipients, such as salts of histidine-HCl, NaCl 2+ or Cl - It may refer to a formulation / pharmaceutical composition that may include.

[0250] With respect to the formulations / pharmaceutical compositions of the present disclosure, the phrases “essentially absent” and “substantially absent” mean that the pharmaceutical composition / formulation contains only trace amounts of an ingredient that are considered negligible for the intended use of the product, that is, the ingredient is present at a level low enough not to affect the performance, stability, safety, or efficacy of the formulation.

[0251] As used herein, the term “binding effect” refers to a characteristic corresponding to a quantitative measure of biological activity (e.g., TTR-binding). A binding effect assay (e.g., ELISA assay) may be used to measure the ability of an anti-TTR antibody or its antigen-binding fragment to trigger a specific response in a disease-related system (e.g., ATTR-CM, e.g., a subject having WT-ATTR-CM). The activity measured in the assay is a surrogate for the intended biological effect and may be used to evaluate the maintenance of that effect over time (e.g., after storage).

[0252] The expressions “can bind” and “bind” as used herein refer to the ability of an antibody or its antigen-binding fragment to bind to agglutinated TTR, for example, under experimental conditions (e.g., in an ELISA assay).

[0253] As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent (e.g., the anti-TTR antibody described herein) combined with one or more pharmaceutically acceptable excipients, diluents, and / or carriers. The pharmaceutical composition is formulated to be administered to a subject, e.g., a mammal, e.g., a human, to prevent, treat, or control a specific disease or condition (e.g., ATTR-CM, e.g., WT-ATTR-CM) that affects or may affect the subject.

[0254] As used herein, the term “pharmaceuticalally acceptable” refers to a compound, substance, composition, and / or form of administration that is within the scope of reasonable medical judgment suitable for use in contact with tissues of a subject, e.g., a mammal (e.g., human), without excessive toxicity, irritation, allergic reaction, or other problems or complications, corresponding to an appropriate benefit / risk ratio.

[0255] The terms "~ to ~" used in this specification include endpoints.

[0256] Room temperature (RT) as used in this specification is defined as 15°C to 25°C according to the European Pharmacopoeia.

[0257] With respect to a reference polynucleotide or polypeptide sequence, the “sequence identity percentage (%)” is defined as the percentage of nucleic acids or amino acids in candidate sequences identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps to achieve the maximum sequence identity percentage as necessary. Alignment to determine % nucleic acid or amino acid sequence identity may be achieved in various ways within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art may determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. For example, % sequence identity values ​​may be generated using the sequence comparison computer program BLAST. As an example, the percentage of sequence identity of a given nucleic acid or amino acid sequence A with respect to a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a specific percentage of sequence identity with respect to a given nucleic acid or amino acid sequence B) is calculated as follows:

[0258] 100 × (X / Y)

[0259] Here, X is the number of nucleotides or amino acids that are scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not the same as the length of nucleic acid or amino acid sequence B, the % sequence identity of A with B will not be the same as the % sequence identity of B with A.

[0260] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and preventive or preventive measures, the purpose of which is to prevent or slow (alleviate) undesirable physiological changes or disorders, such as the development of heart defects. Beneficial or desired clinical outcomes include, without limitation, alleviation of symptoms, reduction in the severity of the disease, a stabilized (i.e., non-deterioration) state of the disease, delay or slowing of disease progression, improvement or temporary remission of the disease state, and remission (partial or total), regardless of whether they are detectable. “Treatment” may also mean extending the survival period compared to the survival period expected without treatment (e.g., extending the survival period of a human subject with ATTR by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years or more, e.g., extending the survival period over the subject’s lifetime). Subjects requiring treatment include not only subjects who already have a disease or disability, but also subjects who are prone to developing a disease or disability or subjects for whom the manifestation of a disease or disability must be prevented.

[0261] The "pH dead band" refers to a small range around the set point where pH fluctuation is allowed before calibration is performed. The "0.05 pH dead band" means that pH fluctuations of ±0.05 units around the target pH are allowed before calibration measures, such as adjusting with acid or base, are applied.

[0262] With respect to polypeptides or antibodies, the term “isolated” refers to molecules recovered from a natural environment, e.g., from a cell culture. “Isolated” antibodies are substantially free of material from the cell source from which they originate. In some embodiments, isolated molecules, e.g., polypeptides, soluble proteins, antibodies, polynucleotides, vectors, and cells, are purified to the extent that they are no longer in the form found in nature. In some embodiments, the isolated molecules are substantially pure. As used herein, the term “substantially pure” refers to a material of at least 50% purity (i.e., free of contaminants), at least 90% purity, at least 95% purity, at least 98% purity, or at least 99% purity. In some embodiments, the purified molecules are pharmaceutical grade.

[0263] The term “polynucleotide” is intended to include not only a single nucleic acid but also multiple nucleic acids, and refers to isolated nucleic acid molecules or constructs, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides may include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds found in peptide nucleic acids (PNA)). The term “nucleic acid” refers to any one or more nucleic acid segments present in a polynucleotide, e.g., DNA or RNA fragments. “Isolated” nucleic acid or polynucleotide means nucleic acid molecules, DNA, or RNA recovered from a natural environment. For example, a recombinant polynucleotide encoding an antibody accommodated in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in xenohost cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules are in vivo or in vitro ( in vitro ) includes RNA transcripts. The isolated polynucleotide or nucleic acid according to the present disclosure further comprises such synthetically produced molecules. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements such as promoters, ribosome binding sites, or transcription terminators. Brief explanation of the drawing

[0264] Fig. 1 : cIEF acidity % of ALXN2220 GMP drug substance batch Fig. 2 : Amount of mannose-containing glycan (Man5) in ALXN2220 GMP drug substance batch. Fig. 3 : iCIEF acid % of small-scale studies. Fig. 4 : Man5 profile of a small-scale study. Fig. 5 : Online pH of a small-scale bioreactor. Fig. 6:Glycosylation profile of ALXN2220 as determined by LCMS. Fig. 7: ATTR deposits in myocardial tissue sections were identified using Amytracker 680, a red fluorescent dye specific to amyloid. ALXN2220 labeled with the green fluorescent dye A488 selectively bound to ATTR, as indicated by the overlap between red and green fluorescence. C. (A ) Refractive index imaging of myocardial tissue sections; ( B ) Confirmation of ATTR deposits in myocardial tissue sections by the red fluorescent dye Amytracker 680; ( C ) Visualization of ALXN2220 labeled with green fluorescent dye A488; ( D The overlap between red and green fluorescence indicates that ALXN2220 binds to ATTR. Fig. 8: ALXN2220 triggered amyloid phagocytosis by macrophages. Dot-like and intracellular red fluorescence patterns indicated the presence of ATTR amyloid within phagocytic vesicles. Macrophages exhibited extensive phagocytic activity, as indicated by the number of red fluorescence vesicles. Fig. ( A ), ( B ), ( C ) and ( D Each represents an exemplary fluorescent image, where the right image is an enlarged section of the left image. Fig. 9 Multinucleated macrophages sequentially detach amyloid fragments from large deposits. Fluorescence images show the fragment detachment process over time (after 2.5 h, 3 h, 4 h, 12 h, 13 h, and 13.5 h). Fig. 10It was observed that macrophages detached thin, elongated ATTR deposits from adjacent cardiomyocytes, moving the deposits more than 20 μm away. Macrophages also cleaved protruding large amyloid deposits, exfoliating small and large fragments of amyloid. Fluorescence images show the migration of deposits over time (after 1 h, 4 h, 5.5 h, 6 h, 11 h, 13.5 h, and 16 h). Specific details for implementing the invention

[0265] In particular, the present specification provides anti-transthyretin (TTR) antibodies, corresponding polynucleotides, and expression vectors, as well as compositions (e.g., pharmaceutical compositions) containing anti-TTR antibodies as drugs and related manufactured articles. Additionally, the present specification provides, in particular, a method for treating or preventing transthyretin-mediated amyloidosis (ATTR) in subjects requiring such treatment or prevention using the pharmaceutical compositions described herein. Furthermore, the present specification provides a method for verifying, identifying, and screening amyloid-depleting drugs using high-resolution live cell imaging, as well as a method for producing pharmaceutical compositions of amyloid-depleting drugs and a quality control method, which includes a kit suitable for use in said method.

[0266] Antibodies and antibody compositions of the present disclosure

[0267] The present disclosure relates to a human-derived monoclonal anti-TTR antibody comprising an immunoglobulin light chain (LC) and an immunoglobulin heavy chain (HC) having the amino acid sequences presented in Table 1 below.

[0268] The above antibody may have an HC having the amino acid sequence of SEQ ID NO. 7 and an LC having the amino acid sequence of SEQ ID NO. 8, wherein,

[0269] X1 is absent, glutamine, or pyroglutamate (pE);

[0270] X2 is methionine or oxidized methionine;

[0271] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide;

[0272] X4 is asparagine or deamidated asparagine;

[0273] X5 is proline or amidated proline;

[0274] X6 is absent or glycine;

[0275] X7 is absent or lysine.

[0276] and LC having the amino acid sequence of sequence number 8.

[0277] In one embodiment, the antibody of the present disclosure comprises HC as presented in SEQ ID NO. 7, wherein glutamine (X1), glycine (X6) and lysine (X7) are present, and the amino acids at positions X2, X3, X4 and X5 are modified (e.g., oxidized, deamidated, containing succinimide, and / or aided) or unmodified as indicated in Table 1.

[0278] In one embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, glycine (X6) and lysine (X7) are present, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0279] In one embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein lysine (X7) is absent, glycine (X6) and glutamine (X1) are present, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0280] In one embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein lysine (X7) and glycine (X6) are absent, glutamine (X1) is present, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0281] In one embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, lysine (X7) is absent, glycine (X6) is present, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0282] In one embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein glutamine (X1) is absent or modified to pyroglutamate, preferably modified to pyroglutamate, lysine (X7) and glycine (X6) are absent, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0283] In a preferred embodiment, the antibody of the present disclosure comprises HC as set forth in SEQ ID NO. 7, wherein glutamine (X1) is modified to pyroglutamate, lysine (X7) is absent, and the amino acids at positions X2, X3, X4 and X5 are modified or unmodified as indicated above.

[0284] In a preferred embodiment, X2, X3, X4, and X5 are unmodified. Accordingly, preferably, the antibody of the present disclosure comprises HC as presented in SEQ ID NO. 7, wherein glutamine (X1) is modified to pyroglutamate, lysine (X7) is absent, and the amino acids at positions X2, X3, X4, and X5 are unmodified.

[0285] The antibody of the present disclosure preferably comprises two HCs each having the amino acid sequence presented in SEQ ID NO. 7 and two LCs each having the amino acid sequence presented in SEQ ID NO. 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated (e.g., one or more amino acids in the HC are N-glycosylated), preferably wherein the N-glycosylated site is located at position Asn300, and the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified.

[0286] In one embodiment, the antibody has an HC having the amino acid sequence of SEQ ID NO. 39 having a PTM as described in detail in the Contents section of the present invention, and an LC having the amino acid sequence of SEQ ID NO. 8. In particular, the antibody may have an HC having the amino acid sequence of SEQ ID NO. 39 and an LC having the amino acid sequence of SEQ ID NO. 8, wherein

[0287] X1 is absent, glutamine or pyroglutamate (pE), preferably pE;

[0288] X2 is methionine or oxidized methionine, preferably methionine;

[0289] X3 is asparagine, deamidated asparagine, or asparagine containing succinimide, preferably asparagine;

[0290] X4 is asparagine or deamidated asparagine, preferably asparagine;

[0291] X5 is proline or amidated proline, preferably proline;

[0292] X6 is absent or glycine, preferably glycine;

[0293] X7 is absent or lysine, preferably absent;

[0294] X I is asparagine or deamidated asparagine, preferably asparagine;

[0295] X II is methionine or oxidized methionine, preferably methionine;

[0296] X III is methionine or oxidized methionine, preferably methionine;

[0297] X IV is aspartate or iso-aspartate, preferably aspartate;

[0298] X V is asparagine or glycosylated asparagine, preferably glycosylated asparagine;

[0299] X VI is methionine or oxidized methionine, preferably methionine;

[0300] X VII It is methionine or oxidized methionine, preferably methionine.

[0301] and LC having the amino acid sequence of sequence number 8.

[0302] The above antibody may have HC having the amino acid sequence of SEQ ID NO. 9 and LC having the amino acid sequence of SEQ ID NO. 8.

[0303] In one embodiment, the heavy chain of the antibody of the present disclosure has lost its C-terminal lysine, that is, the antibody has undergone C-terminal lysine clipping, and the C-terminal lysine is cleaved or absent. In particular, the C-terminal lysine presented in SEQ ID NO. 9 is absent, preferably absent from each heavy chain of the antibody. The sequence, i.e., the sequence of the heavy chain in which the C-terminal lysine is absent, is represented by SEQ ID NO. 10.

[0304] Alternatively, the N-terminal glutamine is modified into pyroglutamate; that is, the heavy chain of the antibody presented in Sequence No. 9 has undergone N-terminal glutaminyl cyclization. The sequence described above, i.e., the sequence of the heavy chain without N-terminal glutamine, is presented in Sequence No. 11. Instead, the N-terminus is cyclized to contain pyroglutamate.

[0305] In a preferred embodiment, the heavy chain of the anti-TTR antibody of the present disclosure has the C-terminal lysine removed as presented in SEQ ID NO. 9, that is, the C-terminal lysine is cleaved or absent, and the N-terminal glutamine is modified to pyroglutamate as presented in SEQ ID NO. 9. The sequence of the heavy chain, i.e., the sequence of the heavy chain without C-terminal lysine and N-terminal glutamine, is presented in SEQ ID NO. 12. Instead, the N-terminus is cyclized and contains pyroglutamate.

[0306] Additionally or alternatively, the antibody is glycosylated, particularly N-glycosylated. More particularly, the heavy chain of the antibody is glycosylated, and even more particularly, the N300 of the heavy chain (sequence number 9) is glycosylated.

[0307] Accordingly, the present disclosure provides antibodies of different species, namely antibodies encoded by the same gene and containing the same primary amino acid sequence but modified post-translationally to different degrees. As mentioned above, the most dominant PTMs are modification of the C-terminus by clipping of lysine and formation of pyroglutamate at the N-terminus, as well as N-glycosylation.

[0308] Furthermore, the theoretical molecular weight of the antibody of the present disclosure is 144.2 kDa, and the weight measured by mass spectrometry (MS) is 144.2 kDa (deglycosylated) and 147.0 to 147.6 kDa (indegradable IgG1), respectively. Accordingly, in one embodiment, the antibody included in the pharmaceutical composition of the present disclosure has a molecular weight of about 150 kDa, preferably about 147 kDa.

[0309] In one embodiment, the antibody of the present disclosure preferably comprises at least eight disulfide crosslinks located at the following positions:

[0310] LC:C23-LC:C88;

[0311] LC:C134-LC:C194;

[0312] LC:C214-HC:C223;

[0313] HC:C22-HC:C97;

[0314] HC:C147-HC:C203;

[0315] HC1:229-HC2:229 and HC1:232-HC2:232;

[0316] HC:C264-HC:C324; and

[0317] HC:C370-HC:C428

[0318] (Here, the numbering of the cysteine ​​residues (C) corresponds to their respective positions in sequence numbers 7 and 8 (wherein sequence number 7 has N-terminal glutamine (X1)) and in sequence numbers 9 and 8 (wherein sequence number 9 has N-terminal glutamine (Q)).

[0319] In one embodiment, the HC of the antibody of the present disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO. 17, wherein the sequence comprising the HC and the signal peptide is presented in SEQ ID NO. 19. Additionally or alternatively, the LC of the antibody of the present disclosure further comprises a signal peptide having the amino acid sequence of SEQ ID NO. 18, wherein the sequence comprising the HC and the signal peptide is presented in SEQ ID NO. 20.

[0320] The present disclosure further relates to a composition comprising any one of the antibody species described above, preferably said antibody species comprising LC as presented in SEQ ID NO. 8 and HC as presented in SEQ ID NO. 7, wherein glutamine (X1) is modified to pyroglutamate, lysine (X7) is absent, amino acids at positions X2, X3, X4 and X5 are unmodified, and X6 is present; or antibody comprising LC as presented in SEQ ID NO. 8 and HC as presented in SEQ ID NO. 9, wherein C-terminal lysine (K) is clipped and N-terminal glutamine is modified to pyroglutamate; or glutamine (X1) is modified to pyroglutamate, lysine (X7) is absent, amino acids at positions X2, X3, X4 and X5 are unmodified, and amino acid X6 is present, preferably asparagine (X V ) is glycosylated, and position X I , X II , X III , X IV , X VI , X VI The amino acid of is an antibody containing HC as presented in sequence number 39, in an unmodified state.

[0321] More preferably, the composition comprises a mixture of the antibody species described above, wherein, preferably, in about 90% to 100% of the antibodies present in the composition (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably about 100% (99.9%)), the N-terminus is modified to pyroglutamic acid, and in about 90% to 100% of the antibodies present in the composition (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, preferably about 96% (95.8%)), the C-terminal lysine is clipped.

[0322] [Table 1]

[0323]

[0324]

[0325]

[0326]

[0327] In a given embodiment, the CDR may contain one or more amino acid residues, particularly in a CDR containing multiple tryptophan (W) residues. These small differences in CDR length may be attributed to the different application of the Martin rule, according to which VHCDR1 terminates immediately before tryptophan (W) and is typically positioned before a W-valine (WV), W-isoleucine (WI), or W-alanine (WA) motif. The heavy chain CDR1 of NI006 / ALXN2220 is sequence 31-SRSSY W G WSince it contains I-39 (Sequence No. 37), the boundary of this CDR may end at Y or G—both of which come before W—depending on how the rule is applied. To avoid misunderstanding, the broadest definition of VHCDR1 containing SRSSY is used herein. In an embodiment, VHCDR1 contains SRSSYWG (Sequence No. 38).

[0328] Polynucleotide and nucleic acid molecules of the present disclosure

[0329] The polynucleotide encoding the antibody of the present disclosure may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. For example, the polynucleotide encoding the antibody may consist of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, DNA and RNA that may be single-stranded or, more typically, double-stranded, or a hybrid molecule comprising a mixture of single-stranded and double-stranded regions. Additionally, the polynucleotide encoding the antibody may consist of a triple-stranded region comprising RNA or DNA or both RNA and DNA. The polynucleotide encoding the antibody may also contain one or more modified bases or DNA or RNA backbones that have been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and non-typical bases, such as inosine. Various modifications may be performed on DNA and RNA; therefore, "polynucleotide" includes forms that have been chemically, enzymatically, or metabolically modified. Isolated polynucleotides encoding non-natural variants of polypeptides derived from immunoglobulins (e.g., immunoglobulin heavy chain portions or light chain portions) may be generated by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin so that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.

[0330] As is well known, RNA can be isolated from original B cells, hybridoma cells, or other transformed cells by standard techniques such as centrifugation or chromatography following guanidinium isothiocyanate extraction and precipitation. If desired, mRNA can be isolated from total RNA by standard techniques such as oligo dT cellulose chromatography. Suitable techniques are familiar in the art. In one embodiment, cDNA encoding the light and heavy chains of an antibody can be prepared simultaneously or separately using reverse transcriptase and DNA polymerase according to well-known methods. PCR can be initiated by consensus constant region primers or by more specific primers based on the disclosed heavy and light chain DNA and amino acid sequences. As discussed above, PCR can also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be screened by consensus primers or greater homology probes, such as human constant region probes.

[0331] DNA, typically plasmid DNA, may be isolated from cells using techniques known in the art, and may be restriction-mapped and sequenced according to standard and well-known techniques, for example, as detailed in the aforementioned references regarding recombinant DNA techniques. Of course, according to the present disclosure, DNA may be synthetic at any point during the isolation process or in subsequent analysis.

[0332] In this regard, the present disclosure also relates to a polynucleotide encoding the antibody of the present disclosure. In particular, the present disclosure relates to a polynucleotide encoding at least one of the immunoglobulin chains of the antibody of the present disclosure.

[0333] In a preferred embodiment of the present disclosure, the polynucleotide comprises, is essentially composed of, or is composed of a nucleic acid having an HC or LC polynucleotide sequence as shown in Table 1.

[0334] Polynucleotides may be generated or manufactured by any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody, for example, [Kutmeier et al. As described in [BioTechniques 17 (1994), 242], it can be assembled from chemically synthesized oligonucleotides, which, in brief, comprises synthesizing overlapping oligonucleotides containing parts of sequences encoding antibodies, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0335] Alternatively, a polynucleotide encoding an antibody or its antigen-binding fragment, variant, or derivative may be generated from nucleic acids from a suitable source. Where a clone containing nucleic acid encoding a specific antibody is not available but the sequence of the antibody molecule is known, the nucleic acid encoding the antibody may be chemically synthesized, or by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a specific gene sequence to identify a cDNA clone from a cDNA library encoding the antibody, for example, an antibody cDNA library isolated from any tissue or cell expressing a TTR-specific antibody, such as a hybridoma cell selected to express the antibody, or a cDNA library generated therefrom, or nucleic acid, preferably polyA +It can be obtained from RNA. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method widely known in the art. Accordingly, in one embodiment of the present disclosure, cDNA encoding an antibody, an immunoglobulin chain, or a fragment thereof is used to generate an anti-TTR antibody.

[0336] In a given embodiment, the polynucleotide or nucleic acid is DNA. In the case of DNA, the polynucleotide containing the nucleic acid encoding the polypeptide may typically include a promoter and / or other transcription or translation control elements operably associated with one or more coding regions. Operable association is when the coding region for a gene product is associated with one or more regulatory sequence(s) in such a manner that the expression of the gene product, e.g., a polypeptide, is placed under the influence or control of the regulatory sequence. Two DNA fragments (e.g., a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" when the induction of the promoter function leads to the transcription of mRNA encoding the desired gene product, and the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequence to induce the expression of the gene product or the ability of the DNA template to be transcribed. Therefore, if the promoter can achieve transcription of the nucleic acid encoding the polypeptide, the promoter region will operably bind to that nucleic acid. The promoter may be a cell-specific promoter that induces substantial transcription of DNA only in predetermined cells. In addition to the promoter, other transcription control elements, such as enhancers, operator factors, repressors, and transcription termination signals, may operably bind to the polynucleotide to induce cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0337] Various transcriptional control regions are known to those skilled in the art. These include, without limitation, transcriptional control regions functioning in vertebrate cells, and these transcriptional control regions include, but are not limited to, promoter and enhancer segments from, for example, cytomegalovirus (with intron-A, the outpost promoter), simian virus 40 (the initial promoter), and retrovirus (e.g., Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock proteins, bovine growth hormone, and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as lymphokine-inducible promoters (e.g., promoters inducible by interferon or interleukin).

[0338] Likewise, various translation control elements are known to those skilled in the art. These include, but are not limited to, ribosomal binding sites, translation initiation and termination codons, and elements derived from picornaviruses (in particular, internal liposomal entry sites (or IRES); which are also referred to as CITE sequences).

[0339] In another embodiment, the polynucleotide of the present disclosure is RNA, for example, in the form of messenger RNA (mRNA).

[0340] The polynucleotide and nucleic acid coding regions of the present disclosure may be combined with additional coding regions encoding a secretion or signal peptide that directs the secretion of the polypeptide encoded by the polynucleotide of the present disclosure. According to the signal hypothesis, proteins secreted by mammalian cells possess a signal peptide or secretion leader sequence, which is cleaved from the mature protein when the growing protein chain begins to be exported across the rough endoplasmic reticulum. Those skilled in the art recognize that polypeptides secreted by vertebrate cells generally possess a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full-length" polypeptide to produce the secreted or "mature" form of the polypeptide. In certain embodiments, a natural signal peptide, e.g., an immunoglobulin heavy or light chain signal peptide, is used, or a functional derivative of the corresponding sequence having the ability to direct the secretion of the polypeptide operably coupled thereto is used. Alternatively, a heterologous mammalian signal peptide or a functional derivative thereof may be used. In a preferred embodiment, a signal peptide as presented in Table 1 is used in connection with the present disclosure.

[0341] Expression of antibody polypeptides

[0342] Polynucleotides encoding antibodies are typically inserted into an expression vector for introduction into host cells that can be used to generate a desired amount of antibody. Once the antibody molecule of the present disclosure or a polynucleotide encoding the heavy or light chain of the antibody is obtained, a vector for generating the antibody molecule can be produced by recombinant DNA technology using techniques well known to those skilled in the art, such as those also described in Attached Example 1. Accordingly, a method for producing a protein by expressing a polynucleotide containing an antibody encoding a nucleotide sequence is described herein. Methods widely known to those skilled in the art may be used to construct an expression vector containing an antibody-coding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present disclosure provides a replicable vector comprising a nucleotide sequence encoding the antibody molecule of the present disclosure or its heavy or light chain, which is operably linked to a promoter. Such a vector may include a nucleotide sequence encoding an invariant region of an antibody molecule, and the variable domain of the antibody may be cloned into such a vector for the expression of the entire heavy chain or light chain.

[0343] In this specification, the terms “vector” or “expression vector” refer to a vector used in accordance with this disclosure as a carrier for introducing and expressing a desired gene into a host cell. As is known to those skilled in the art, such a vector may be selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, a vector suitable for this disclosure will include a selection marker, a suitable restriction site to facilitate the cloning of the desired gene, and the ability to enter and / or replicate into a eukaryotic or prokaryotic cell. For the purposes of this disclosure, a number of expression vector systems may be used. For example, one class of vectors utilizes DNA sequences derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others include the use of polycistronic systems having internal ribosome binding sites. Additionally, cells in which DNA has been integrated into the chromosome can be selected by introducing one or more markers that enable the selection of transfected host cells. The markers may provide prototrophy for trophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for the optimal synthesis of mRNA. These elements may include signal sequences and splice signals, as well as transcription promoters, enhancers, and termination signals as also described in Attached Example 1.

[0344] In a particularly preferred embodiment, the cloned light and heavy chain genes are inserted into an expression vector along with the signal peptide sequences as described above, preferably into two different expression vectors (one for the heavy chain gene and one for the light chain gene). Examples of suitable vectors include, but are not limited to, plasmids pcDNA3, pHCMV / Zeo, pCR3.1, pEF1 / His, pIND / GS, pRc / HCMV2, pSV40 / Zeo2, pTRACER-HCMV, pUB6 / V5-His, pVAX1, and pZeoSV2 (available from Invitrogen, San Diego, California, USA), and plasmid pCI (available from Promega, Madison, Wisconsin, USA). Generally, screening a number of transformed cells suitablely expressing high levels of immunoglobulin heavy and light chains is a routine experiment that can be performed, for example, by a robotic system. Vector systems are also disclosed in U.S. Patents No. 5,736,137 and No. 5,658,570, each incorporated herein by reference in its entirety. These systems provide high expression levels, e.g., > 30 pg / cell / day. Other exemplary vector systems are disclosed, e.g., in U.S. Patent No. 6,413,777.

[0345] In another preferred embodiment, the antibody of the present disclosure or its antigen-binding fragment, variant, or derivative may be expressed using a polycistronic construct such as that disclosed in U.S. Patent Application Publication No. 2003-0157641 A1 and incorporated herein in its entirety. In such an expression system, multiple gene products of interest, such as the heavy and light chains of the antibody, may be generated from a single polycistronic construct. Such a system advantageously uses an internal liposomal entry site (IRES) to provide relatively high levels of the antibody. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980, which is also incorporated herein. Those skilled in the art will understand that such an expression system may be used to effectively generate the full range of antibodies disclosed in this application. Accordingly, in one embodiment, the present disclosure provides a vector comprising a polynucleotide encoding a binding domain or variable region of at least one immunoglobulin chain of an antibody, optionally combined with a polynucleotide encoding a variable region of the remaining other immunoglobulin chain of said binding molecule.

[0346] More generally, once a vector or DNA sequence encoding a monomeric subunit of an antibody is prepared, the expression vector can be introduced into a suitable host cell. Introducing a plasmid into a host cell can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection—including, for example, lipotransfection using Fugene® or Lipofectamine—protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection by an intact virus. Typically, plasmid introduction into a host is performed via a standard calcium phosphate co-precipitation method. Host cells harboring the expression construct are grown under conditions suitable for the production of light and heavy chains and are tested for heavy and / or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting assay (FACS), and immunohistochemistry.

[0347] The expression vector is delivered to a host cell by conventional techniques, and the transfected cell is then cultured by conventional techniques to produce an antibody for use in the method described herein. Accordingly, the present disclosure comprises a host cell comprising an antibody of the present disclosure or a polynucleotide encoding a heavy chain or a light chain thereof, which is preferably operably linked to a heterologous promoter. Additionally or alternatively, the present disclosure also comprises a host cell comprising a vector as defined above, comprising a polynucleotide encoding at least a binding domain or variable region of the immunoglobulin chain of the antibody, in combination with a polynucleotide encoding a variable region of the remaining other immunoglobulin chain of said binding molecule. In one embodiment for the expression of a double-chain antibody, as described in detail below, a single vector or vectors encoding both the heavy chain and the light chain may be co-expressed in the host cell for the expression of the entire immunoglobulin molecule.

[0348] Host cells may be co-transfected with two expression vectors of the present disclosure, which are a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker that enables the same expression of the heavy chain and light chain polypeptides. Alternatively, a single vector encoding both heavy chain and light chain polypeptides may be used. In such circumstances, the light chain is advantageously positioned before the heavy chain to avoid an excess of toxic free heavy chains; [Proudfoot, Nature 322 (1986), 52]; [Kohler, Proc. Natl. Acad. Sci. USA 77] See (1980), 2197). The coding sequences for the heavy and light chains may include cDNA or genomic DNA.

[0349] As used herein, "host cell" refers to a cell constructed using recombinant DNA techniques and possessing a vector encoding at least one heterogeneous gene. In the description of the process for isolating antibodies from a recombinant host, the terms "cell" and "cell culture medium" are used interchangeably to denote the source of the antibodies unless explicitly stated otherwise. In other words, recovery of polypeptides from "cells" may mean recovery from spin-down whole cells or recovery from a cell culture medium containing both a medium and suspended cells.

[0350] Various host-expression vector systems may be used to express antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles in which the coding sequence of interest can be generated and subsequently purified, but also represent cells capable of expressing the antibody molecules of the present disclosure in the same reaction system when transformed or transfected with an appropriate nucleotide coding sequence. These are bacteria (e.g., Escherichia coli) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing an antibody coding sequence. Escherichia coli ) and Bacillus subtilis ( Bacillus subtilis )); yeast transformed with a recombinant yeast expression vector containing an antibody-coding sequence (e.g., Saccharomyces pichia ( Saccharomyces pichiaInsect cell systems infected with a recombinant viral expression vector containing an antibody-coding sequence (e.g., baculovirus); plant cell systems infected with a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector containing an antibody-coding sequence (e.g., Ti plasmid); or mammalian cell systems (e.g., COS, CHO, NSO, BLK, 293, 3T3 cells) possessing a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter), but not limited thereto. Preferably, bacterial cells, e.g., E. coli, more preferably eukaryotic cells are used for the expression of the recombinant antibody molecule, particularly for the expression of the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, along with vectors such as major intermediate early gene promoter elements from human cytomegalovirus, are effective expression systems for antibodies. For example, literature [Foecking et al. , Gene 45 (1986), 101]; literature[Cockett et al. See , Bio / Technology 8 (1990), 2].

[0351] Host cell lines used for protein expression are often of mammalian origin; and those skilled in the art are recognized as having the ability to preferentially determine the specific host cell line most suitable for the expression of the desired gene product. Exemplary host cell lines include, but are not limited to, CHO (Chinese hamster ovary), DG44 and DUXB11 (Chinese hamster ovary cell lines, DHFR deficient), HELA (human cervical carcinoma), CVI (monkey kidney cell line), COS (derived of CVI with SV40 T antigen), VERY, BHK (puppy hamster kidney), MDCK, WI38, R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). CHO and 293 cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection (ATCC), or published literature.

[0352] In addition, a host cell strain may be selected to regulate the expression of the inserted sequence or to modify and process the gene product in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells possess characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. An appropriate cell line or host system may be selected to ensure the proper modification and processing of the expressed foreign protein. To this end, eukaryotic host cells possessing cellular mechanisms for the proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0353] In a preferred embodiment, CHO cells, most preferably CHO cell line K1 as shown in Example 1, are used to express the polynucleotide of the present disclosure to produce an antibody and an immunoglobulin chain, respectively.

[0354] For the long-term, high-yield production of recombinant proteins, stable expression is desirable. For example, cell lines that stably express antibody molecules can be engineered. Rather than using an expression vector containing a viral replication origin, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. After the introduction of foreign DNA, the engineered cells can be grown in enriched medium for 1 to 2 days and then switched to selective medium. Selectable markers in the recombinant plasmid confer resistance to selection, causing the cells to grow to stably incorporate the plasmid into their chromosomes and form a focus that can be subsequently cloned and expanded into a cell line. This method can advantageously be used to engineer cell lines that stably express antibody molecules.

[0355] Herpes simplex virus thymidine kinase (Literature [Wigler] et al. , Cell 11 (1977), 223]), hypoxanthine guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48:1992, 202]) and adenine phosphoribosyltransferase (Lowy et al. A number of screening systems, including but not limited to [ , Cell 22 (1980), 817]), may be used, and the genes may be used in tk-, hgprt-, or aprt- cells, respectively. Additionally, antimetabolite resistance may be used as a basis for screening for the following genes: dhfr, which confers resistance to methotrexate (reference [Wigler et al. , Natl. Acad. Sci. USA, 77:1980, 357]; literature[O'Hare et al. , Proc. Natl. Acad. Sci. USA 78 (1981), 1527]); gpt conferring resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78 (1981), 2072]); neo conferring resistance to aminoglycoside G-418 (Goldspiel et al. , Clinical Pharmacy 12 (1993), 488-505]; literature [Wu and Wu, Biotherapy 3 (1991), 87-95]; literature [Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 (1993), 573-596]; literature [Mulligan, Science 260 (1993), 926-932]; and literature [Morgan and Anderson, Ann. Rev. Biochem. 62 (1993), 191-217]; literature [TIB TECH 11 (1993), 155-215]; and hygro (which confers resistance to hygromycin) literature [Santerre et al. Methods of recombinant DNA technology that may be used and are generally known in the art are described in the literature [Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993)]; in the literature [Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990)]; and in the literature [Chapters 12 and 13, Dracopoli et al.(eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994)]; Colberre-Garapin et al. [J. Mol. Biol. 150:1 (1981)] is described, and these are incorporated herein by reference in their entirety.

[0356] In vitro production enables scaling up to provide the desired polypeptide in large quantities. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or a continuous stirring reactor, or culture of immobilized or captured cells, e.g., in hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the polypeptide solution may be purified by conventional chromatographic methods, e.g., gel filtration, ion-exchange chromatography, chromatography on DEAE-cellulose, or (immuno-)affinity chromatography, e.g., after the preferential biosynthesis of the synthetic hinge region polypeptide or before or after the HIC chromatography step described herein.

[0357] Genes encoding the antibodies of the present disclosure, or their antigen-binding fragments, variants, or derivatives, may also be expressed in bacteria or non-mammalian cells such as insects, yeast, or plant cells. Bacteria that readily absorb nucleic acids include the enterobacteriaceae, such as E. coli or Salmonella ( Salmonella ) strains; Bacillaceae, e.g., B. subtilis ( B. subtilis ); Pneumococcus( Pneumococcus ); Streptococcus( Streptococcus ); and Haimophilus influenzae( Haemophilus influenzaeMembers of ). It will be further understood that when expressed in bacteria, the heteropeptide typically becomes part of an inclusion body. The heteropeptide must be isolated, purified, and then assembled into a functional molecule. If a quadrivalent antibody is required, the subunits will be self-assembled into a quadrivalent antibody. For example, refer to International Application WO 02 / 096948.

[0358] In bacterial systems, multiple expression vectors can be advantageously selected depending on the intended use of the antibody molecule being expressed. For example, if large quantities of such proteins need to be produced, a vector directing the expression of high levels of easily purified fusion protein products may be desirable for the production of pharmaceutical compositions of antibody molecules. Such a vector is the E. coli expression vector pUR278 (Ruther et al. [Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109]; [Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503-5509]); pIN vectors (including, but not limited to, [Inouye and Inouye, Nucleic Acids Res. 13 (1985), 3101-3109]; [Van Heeke and Schuster, J. Biol. Chem. 24 (1989), 5503-5509]). pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a matrix of glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

[0359] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae ( SSaccharomyces cerevisiae ) or general baking yeast is the most commonly used among eukaryotic microorganisms, but many other strains are generally available, for example, Pichia pastoris ( Pichia pastoris There is ). For expression in Saccharomyces, for example, plasmid YRp7 (literature [Stinchcomb et al. , Nature 282 (1979), 39]; literature[Kingsman et al. , Gene 7 (1979), 141]; literature[Tschemper et al. [Jones, Genetics 10 (1980), 157]) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for mutant strains of yeast that cannot grow on tryptophan (e.g., ATCC No. 44076 or PEP4-1) (Jones, Genetics 85 (1977), 12]). Subsequently, the presence of trpl deletion as a feature of the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0360] In insect systems, Autographa Californica ( Autographa californica ) Nuclear polyhedral disease virus (AcNPV) is typically used as a vector to express foreign genes. The virus is Spodoptera prugifera ( Spodoptera frugiperda It grows in cells. The antibody-coding sequence can be individually cloned into a non-essential region of the virus (e.g., polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., polyhedrin promoter).

[0361] Once the antibody molecule of the present disclosure has been recombinantly expressed, the whole antibody, its dimer, individual light and heavy chains, or other immunoglobulin forms of the present disclosure may be purified according to standard procedures in the art, including, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly, by affinity for a specific antigen following protein A chromatography), size determination column chromatography, and kappa selective affinity chromatography), centrifugation, by differential solubility (e.g., ammonium sulfate precipitation), or by any other standard technique for the purification of proteins; see, for example, the literature [Scopes, "Protein Purification", Springer Verlag, NY (1982)]. Alternatively, a preferred method for increasing the affinity of the antibody of the present disclosure is disclosed in U.S. Patent Publication No. 2002-0123057 A1. Accordingly, in one embodiment, the present disclosure also provides a method for producing an anti-TTR antibody or an antibody that recognizes a mutated, misfolded, misassembled, or aggregated TTR species and / or a fragment thereof or an immunoglobulin chain(s) thereof, said method

[0362] (a) a step of culturing a host cell as defined above - said cell comprises a polynucleotide or vector as defined above -; and

[0363] (b) a step of isolating the antibody or its immunoglobulin chain(s) from the culture medium.

[0364] Furthermore, in one embodiment, the present disclosure also relates to an antibody or its immunoglobulin chain(s) obtainable by the method of preparing an antibody encoded by a polynucleotide as defined above, or an anti-TTR antibody or an anti-TTR antibody or a mutated, misfolded, misassembled, or aggregated TTR species and / or fragments thereof or its immunoglobulin chain(s).

[0365] Pharmaceutical composition / formulation

[0366] The present disclosure provides a pharmaceutical composition each containing an anti-TTR antibody as defined above and a mixture of anti-TTR antibody species.

[0367] The pharmaceutical composition of the present disclosure may be formulated as described below. For example, a pharmaceutical composition containing an anti-TTR antibody may be formulated to include sucrose, polysorbate 80, and histidine. Furthermore, a pharmaceutical composition containing an anti-TTR antibody may be formulated at a desired pH (e.g., pH 5.8) as described herein. A pharmaceutical composition containing an anti-TTR antibody may further include pharmaceutically acceptable excipients or diluents as described herein.

[0368] For example, a pharmaceutical composition may comprise an anti-TTR antibody of the present disclosure at a concentration of about 50 mg / mL in a volume of 2.0 mL, a histidine buffer with a pH of about 5.8, 6.5% w / v sucrose, and 0.03% w / v polysorbate 80.

[0369] In another example, the pharmaceutical composition may comprise an anti-TTR antibody of the present disclosure at a concentration of about 50 mg / mL in a total volume of 2.0 mL, a histidine buffer with a pH of about 5.8, 8.0% w / v sucrose, and 0.03% w / v polysorbate 80.

[0370] In a preferred embodiment, the antibody comprises an HC chain having the amino acid sequence presented in SEQ ID NO. 7, 39 or 9, and an LC having the amino acid sequence presented in SEQ ID NO. 8.

[0371] As shown in Example 2, about 99% to 100% of the antibodies present in the pharmaceutical formulation have N-terminal pyro-glutamic acid in the heavy chain, and about 96% of the antibodies have C-terminal lysine loss. Therefore, in one embodiment, about 99% of the antibodies in the formulation of the present disclosure have a heavy chain in which N-terminal pyro-glutamic acid is modified from N-terminal glutamine, and / or about 96% of the antibodies have C-terminal lysine loss.

[0372] In a preferred embodiment, the antibody of the present disclosure consists of two HCs having the amino acid sequence presented in SEQ ID NO. 7 and two LCs having the amino acid sequence presented in SEQ ID NO. 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is present at position Asn300, and wherein the amino acids at positions X2, X3, X4, and X5 are modified or unmodified, preferably unmodified. In a further preferred embodiment, the antibody of the present disclosure comprises two HCs having the amino acid sequence presented in SEQ ID NO. 39 and two LCs having the amino acid sequence presented in SEQ ID NO. 8, wherein in each HC, the N-terminal glutamine (X1) is modified to pyroglutamic acid, the C-terminal lysine (X7) is absent, and the heavy chain is N-glycosylated, preferably wherein the N-glycosylated site is at position Asn300 (X V Exists at ), where the amino acids at positions X2, X3, X4, and X5 are modified or undeformed, preferably undeformed, and at position X I , X II , X III , X IV, X VI , X VI The amino acids are modified or unmodified, preferably unmodified.

[0373] In a further preferred embodiment, the antibody of the present disclosure comprises two HCs having SEQ ID NO. 9 and two LCs having SEQ ID NO. 8, wherein the N-terminal glutamine in the HCs is modified to pyroglutamic acid and the C-terminal lysine is eliminated, i.e., the C-terminal lysine is removed or absent, and the heavy chain is N-glycosylated, preferably, wherein the N-glycosylated site is located at position Asn300. In other words, the antibody comprises two heavy chains having SEQ ID NO. 12 (preferably the N-terminus is cyclized) and two light chains having SEQ ID NO. 8, wherein the heavy chain is N-glycosylated, preferably, wherein the N-glycosylated site is located at position Asn300.

[0374] In addition, to a negligible degree and preferably in negligible amounts, some antibody species may be found in the analyzed antibody compositions that 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 referred to in Examples 2 and 3. In particular, following the C-terminal lysine clipping and N-terminal cyclization mentioned above, the antibody may exhibit methionine (M) oxidation at position HC 255; asparagine (N) deamidation at positions HC 318 and / or HC 387; asparagine (N) succinimide formation at position HC 318; and / or amidation of C-terminal proline (P) following the loss of C-terminal lysine and glycine.

[0375] antibody concentration

[0376] The antibody of the present disclosure may be formulated into a pharmaceutical composition described herein in which the concentration of the anti-TTR antibody of the present disclosure is about 25 mg / mL to about 150 mg / mL (e.g., about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, or about 125 mg / mL), preferably about 50 mg / mL to about 100 mg / mL (e.g., about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL), most preferably 50 mg / mL or 100 mg / mL.

[0377] Sucrose

[0378] The pharmaceutical composition also comprises an amount of sucrose, for example, about 6% to about 9%, about 6% to about 7%, or about 7.5% to about 8.5% by weight / unit volume (w / v) (e.g., about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, or 9% w / v of sucrose). For example, the pharmaceutical composition may contain about 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.45%, 6.5%, 6.55%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.05%, 8.1%, 8.2%, 8.3%, 8.4%, or 8.5% w / v sucrose. In particular, the pharmaceutical composition contains about 6.5% w / v sucrose or about 8% w / v sucrose, most preferably 8% w / v sucrose.

[0379] Polysorbate

[0380] The pharmaceutical composition also comprises, for example, an amount of polysorbate of about 0.001% to about 0.1% w / v (e.g., about 0.001%, 0.005%, 0.01%, 0.05% or 0.1% w / v PS (80)), for example, polysorbate 20 or polysorbate 80, preferably polysorbate 80 (PS80). For example, the pharmaceutical composition may contain about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% w / v PS80. In particular, the pharmaceutical composition contains about 0.03% w / v PS(80).

[0381] buffer

[0382] The pharmaceutical composition also comprises a histidine buffer. The pharmaceutical composition may comprise the buffer in an amount of, for example, about 1 mM to about 100 mM (e.g., about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM). For example, the pharmaceutical composition may comprise 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, approx. 21 mM, approx. 22 mM, approx. 23 mM, approx. 24 mM, approx. 25 mM, approx. 26 mM, approx. 27 mM, approx. 28 mM, approx. 29 mM, approx. 30 mM, approx. 31 mM, approx. 32 mM, approx. 33 mM, approx. 34 mM, approx. 35 mM, approx. 36 mM, approx. 37 mM, approx. 38 mM, approx. 39 mM, approx. 40 mM, approx. 41 mM, approx. 42 mM, approx. 43 mM, approx. 44 mM, approx. 45 mM, approx. 46 mM, approx. 47 mM, approx. 48 mM, approx. 49 mM, approx. 50 mM, approx. 51 mM, approx. 52 mM, approx. 53 mM, approx. 54 mM, approx. 55 mM, approx. 56 mM, approx. 57 mM, approx. 58 mM, approx. 59 mM, approx. 60 mM, approx. It may contain 61 mM, approximately 62 mM, approximately 63 mM, approximately 64 mM, approximately 65 mM, approximately 66 mM, approximately 67 mM, approximately 68 mM, approximately 69 mM, approximately 70 mM, approximately 71 mM, approximately 72 mM, approximately 73 mM, approximately 74 mM, approximately 75 mM, approximately 76 mM, approximately 77 mM, approximately 78 mM, approximately 79 mM, approximately 80 mM, approximately 81 mM, approximately 82 mM, approximately 83 mM, approximately 84 mM, approximately 85 mM, approximately 86 mM, approximately 87 mM, approximately 88 mM, approximately 89 mM, approximately 90 mM, approximately 91 mM, approximately 92 mM, approximately 93 mM, approximately 94 mM, approximately 95 mM, approximately 96 mM, approximately 97 mM, approximately 98 mM, approximately 99 mM, or approximately 100 mM. In particular, the pharmaceutical composition comprises about 20 mM histidine, preferably L-histidine and L-histidine monohydrochloride.

[0383] pH

[0384] The pharmaceutical composition may have a pH of about 5.0 to about 8.0 (e.g., about 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). For example, the pharmaceutical composition may have a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.75, 5.8, 5.85, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In particular, the pharmaceutical composition has a pH of about 5.8.

[0385] volume

[0386] The pharmaceutical composition is about 1 mL to about 100 mL, about 1 mL to about 50 mL, about 5 mL to about 25 mL, about 20 mL to about 25 mL, or about 1 mL to about 10 mL (e.g., about 1 mL to about 80 mL, about 1 mL to about 70 mL, about 1 mL to about 60 mL, about 1 mL to about 50 mL, about 1 mL to about 40 mL, about 1 mL to about 30 mL, about 1 mL to about 20 mL, about 1 mL to about 10 mL, about 5 mL to about 20 mL, about 5 mL to about 15 mL, about 5 mL to about 10 mL, about 10 mL to about 20 mL, about 15 mL to about 20 mL, about 20 mL to about 22.5 mL, about 20 mL to about 25 mL, about 1 mL to about 9 It may be provided in volumes of about 1 mL to about 8 mL, about 1 mL to about 7 mL, about 1 mL to about 6 mL, about 1 mL to about 5 mL, about 1 mL to about 4 mL, about 1 mL to about 3 mL, about 1 mL to about 2.25 mL, about 1 mL to about 2 mL, or about 2 mL to about 2.25 mL (e.g., in a vial or other container as described herein). For example, a pharmaceutical composition is about 1 mL to about 2.25 mL (e.g., about 1 mL to about 2.2 mL, about 1 mL to about 2 mL, about 1 mL to about 1.8 mL, about 1 mL to about 1.6 mL, about 1 mL to about 1.4 mL, about 1 mL to about 1.2 mL, about 1.5 mL to about 1.25 mL, about 1.5 mL to about 2 mL, about 1.9 mL to about 1.2 mL, about 2.1 mL to about 2.25 mL) or about 1 mL to about 100 mL (e.g., about 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.It may exist in volumes of 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.

[0387] In a 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.

[0388] Additional formulations

[0389] The pharmaceutical composition may be prepared such that it is not reconstituted from the lyophilized anti-TTR antibody and / or is not further lyophilized. Additionally, the pharmaceutical composition may be prepared such that it is essentially free of sodium chloride and / or essentially free of poloxamer. The pharmaceutical composition may also be prepared as a sterile composition.

[0390] In the most preferred embodiment of the present disclosure, the pharmaceutical composition comprises or consists of 50 mg / mL of the antibody of the present disclosure, 20 mM histidine (L-histidine 1.06 mg / mL and L-histidine monohydrochloride 2.78 mg / mL), 65 mg / mL or 80 mg / mL of sucrose, preferably 80 mg / mL of sucrose, 0.3 mg / mL of polysorbate 80, and a water for infusion / injection at pH 5.8.

[0391] The disclosure in PCT Publication WO2024105092 A1 is incorporated herein by reference in all relevant parts.

[0392] characteristic

[0393] The present disclosure features the invention of a pharmaceutical composition having enhanced features (e.g., stability, solubility, storage, etc.) as described in this specification (e.g., see Example 4). The pharmaceutical composition may preferably be characterized by having a shelf life of 24 months at 2°C to 8°C when protected from light.

[0394] The pharmaceutical composition of the present disclosure may be characterized by one, two, three, or all four of the following stability criteria (i) to (iv) in any combination: (i) a main peak reduction (indicating monomer content) under heat stress conditions during long-term storage at about 40°C for 1 month, at about 25°C for 6 months, or at about 5°C for 18 months, when measured by SEC-HPLC, less than 5%, preferably less than 4%, more preferably less than 3%, more preferably less than 2%; (ii) the pharmaceutical composition exhibits an acidic species content of the anti-TTR antibody or its antigen-binding fragment of about 40% or less when measured by cIEF under heat stress conditions at about 40°C for 2 weeks or at about 25°C for 3 months; (iii) the pharmaceutical composition shows that the content of acidic species of the anti-TTR antibody or its antigen-binding fragment is less than 40%, preferably less than 35%, when measured by cIEF under long-term storage conditions of about 5°C for 12 or 18 months; and / or (iv) the anti-TTR antibody or its antigen-binding fragment maintains at least 80%, preferably at least 90%, of the binding efficacy to TTR protein when measured by ELISA after storage at about 25°C for 6 months, or after storage at about 5°C for 12 or 18 months, compared to the control group (e.g., no extended storage).

[0395] Preferably, the pharmaceutical composition exhibits any or all of the features as presented in the table of Example 4.

[0396] In particular, the pharmaceutical composition has an osmolal concentration of ≥ 240 mOsm / Kg.

[0397] The pharmaceutical composition is stable upon freezing and thawing. As used in relation to the pharmaceutical compositions described herein, the terms “stable” or “stability” as used herein refer to the maintenance of the physical and functional characteristics of the composition over time. For example, a stable composition may be described as maintaining appearance (e.g., color, milky white, number of visible particles, and / or number of subvisible particles), pH, antibody concentration, and / or osmolarity after long-term storage.

[0398] The pharmaceutical composition of the present disclosure is presented as remaining stable for at least 1 month at 40±2°C and 75±5% RH (stress stability study); for at least 6 months at 25±2°C / 60±5% RH (accelerated stability study); and for at least 12 to 18 months at 5±3°C (long-term stability study). Furthermore, the extinction coefficient of the pharmaceutical composition is 1.438 (mg / mL). -1 cm -1 It was determined that the pharmaceutical formulation is a sterile, colorless to slightly yellowish, transparent to slightly milky white solution, and was essentially presented as having no visible particles at a pH of 5.8.

[0399] Treatment methods

[0400] A composition of the present invention containing the antibody of the present disclosure, the polynucleotide(s) of the present disclosure, the vector(s) of the present disclosure, the host cell of the present disclosure, or the anti-TTR antibody of the present disclosure may be used for the prophylactic or therapeutic treatment of diseases associated with TTR amyloidosis, e.g., ATTR, e.g., ATTR-CM, ATTR polyneuropathy (ATTR-PN), familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), senile systemic amyloidosis (SSA), systemic familial amyloidosis, meningeal / central nervous system (CNS) amyloidosis including Alzheimer's disease, TTR-associated ocular amyloidosis, TTR-associated renal amyloidosis, TTR-associated hyperthyroxinemia, carpal tunnel syndrome, TTR-associated ligament amyloidosis including rotator cuff tear and lumbar spinal stenosis, and preeclampsia. Furthermore, the antibody of the present disclosure may be used for in vivo detection or imaging of TTR in humans or animals, or as a therapeutic and / or diagnostic agent targeting TTR, and preferably, said in vivo imaging includes scintigraphy, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR), optical imaging, or magnetic resonance imaging (MRI).

[0401] In particular, the pharmaceutical compositions described herein may be used in a method for treating or preventing transthyretin-mediated amyloidosis (ATTR). Additionally, the pharmaceutical compositions described herein may be used in a method for treating or preventing ATTR amyloidosis associated with cardiomyopathy (ATTR-CM, e.g., WT-ATTR-CM). For example, a pharmaceutical composition containing the human anti-TTR antibody of the present disclosure at a concentration of about 50 mg / mL, histidine buffer with a pH of about 5.8, 6.5% or 8.0% w / v sucrose, and 0.03% w / v polysorbate 80 may be administered to treat ATTR or ATTR-CM, e.g., WT-ATTR-CM.

[0402] A pharmaceutical composition containing an anti-TTR antibody may be administered to human subjects to treat, prevent, or control transthyretin-mediated amyloidosis (ATTR), e.g., ATTR amyloidosis accompanied by cardiomyopathy (ATTR-CM, e.g., WT-ATTR-CM). The pharmaceutical composition is preferably administered by intravenous injection or infusion.

[0403] The compositions and methods provided herein may be used to treat subjects having 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 thyroidemia, TTR-related ligament amyloidosis, carpal tunnel syndrome, rotator cuff tear, lumbar stenosis, preeclampsia, or known pathogenic TTR mutations (e.g., those causing amyloidosis). Subjects may have sporadic, WT-ATTR-CM, and negative genetic test results for TTR mutations.

[0404] The pharmaceutical composition can be immediately used to administer to a subject requiring it, preferably via intravenous infusion. The pharmaceutical composition may be diluted with glucose or a polymer thereof before infusion, preferably the polymer is dextran. The concentration of glucose or its polymer may be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% w / v, preferably 5% glucose.

[0405] courage

[0406] A container comprising the pharmaceutical composition of the present disclosure is also provided herein. Suitable containers include, for example, bottles (e.g., infusion bottles), vials (e.g., Type I clear glass vials), syringes, IV solution bags, etc. The container may be formed from various materials such as glass or plastic. The container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper that can be punctured by a subcutaneous injection needle).

[0407] Containers such as type I clear glass vials or injection bottles may be sized at 1 mL, 2 mL, 5 mL, 10 mL, 15 mL, 20 mL, or 25 mL and may accommodate a pharmaceutical composition with a volume overfill of about 10% to about 15% (e.g., 10% or 12.5%). The container may contain the pharmaceutical composition described in this specification in a predetermined volume, such as, for example, 0.5 mL to 10 mL, 2 mL to 2.25 mL, 10 mL to 20 mL, 15 mL to 25 mL, 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 pharmaceutical composition, i.e., the drug product described herein, is presented as a sterile, colorless to slightly yellowish, transparent to slightly milky liquid, essentially free of visible particles, and is provided as a concentrate of an infusion solution supplied in a 2 mL (2R) glass vial having an aluminum flip-off cap over a 13 mm rubber stopper. The product is preferably diluted in sterile glucose before administration, which is a commercial product and is not attached to the drug product in one embodiment. Preferably, the container is a 2 mL or 20 mL vial, most preferably a glass vial having an aluminum flip-off cap over a 13 mm rubber stopper, which preferably accommodates a pharmaceutical formulation with approximately 12.5% ​​volume overfill, or a total volume of 2.25 mL or 22.5 mL.

[0408] Manufactured goods / treatment kits

[0409] The present disclosure also provides a manufactured article (e.g., a kit, particularly a therapeutic kit) containing a substance useful for the treatment or prevention of transthyretin-mediated amyloidosis (ATTR) in human subjects. The manufactured article comprises one or more of the containers of the present disclosure as described above (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 50 or more) and a label or package insert on or associated with one or more of such containers, wherein the container comprises a composition of the present disclosure.

[0410] A label or package insert indicates that the composition is used to treat ATTR. The manufactured article may comprise, at least, a first container containing the pharmaceutical composition. Optionally, the manufactured article may further comprise a second container containing a second therapeutic agent, such as a TTR tetramer stabilizer (e.g., tafamidis). The manufactured article in this embodiment of the disclosure may further comprise a package insert indicating that the composition can be used to treat ATTR. Alternatively or additionally, the manufactured article may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as BWFI, PBS, Ringer's solution, and dextrose solution.

[0411] The manufactured article may additionally include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. For example, the manufactured article (e.g., kit) may include a second container containing a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or a polymer thereof, such as glucose or dextran (e.g., at a concentration of about 5% w / v). The manufactured article may include other materials, including a package insert containing a buffer, diluent, filter, needle, syringe, and instructions for use.

[0412] In a preferred embodiment, the manufactured article comprises a vial, preferably a clear glass vial sealed with a (gray) rubber stopper and a (blue) aluminum-plastic cover flip-off cap. Preferably, the antibody is presented in the vial at a concentration of 50 mg / mL and is presented as a sterile, colorless to slightly yellowish, clear to slightly milky liquid, and is provided as a concentrate of an infusion solution that is essentially free of visible particles. In a further preferred embodiment, the manufactured article comprises a dosing syringe in a dosing pump or infusion bag containing the diluted antibody formulation.

[0413] Live cell imaging method of the present disclosure

[0414] Based on the results obtained during experiments conducted within the scope of the present invention, a method for screening, verifying, characterizing, and quality controlling amyloid-depleting drugs has been developed.

[0415] In particular, a method for verifying an amyloid-depleting drug, a screening method for identifying and selectively acquiring an amyloid-depleting drug from a plurality of test compounds, a method for analyzing the effect of an agent, e.g., a second drug, on the amyloid-depleting activity of the amyloid-depleting drug, and a screening method for the ability of an amyloid-depleting drug to bind to amyloid, mediate macrophage recruitment to amyloid deposits, and then perform amyloid fragmentation and internalization, all comprise the steps of incubating a tissue section containing amyloid deposits with macrophages in the presence of an amyloid-depleting drug or, in the case of a screening method, a test compound, and performing high-resolution live cell imaging. Live cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. Thus, the tissue section is stained with an amyloid-specific fluorescent dye, and as a result, the amyloid deposits within the tissue section are stained; The amyloid-depleting drug and the test compound are each labeled with a fluorescent dye, and as a result, when the amyloid-depleting drug and the test compound bind to amyloid, the amyloid deposits within the tissue section are labeled; or the tissue section is stained with an amyloid-specific dye, and the amyloid-depleting drug and the test compound are each labeled.

[0416] In one embodiment, a tissue section is stained with an amyloid-specific fluorescent dye before the addition of macrophages and an amyloid-depleting drug (or a test compound in the case of a screening method), and the amyloid-depleting drug and the test compound are each labeled with a fluorescent dye, and this approach is preferably used to visualize and detect the binding of each of the amyloid-depleting drug and the test compound to amyloid deposits by generating a superimposed image of the fluorescent signals of each of the amyloid-depleting drug and the test compound and the fluorescent signal emitted by the amyloid-specific fluorescent dye. In this embodiment, each of the amyloid-depleting drug and the test compound is labeled with a fluorescent dye different from the fluorescent dye used to stain the amyloid deposits.

[0417] In one embodiment, tissue sections are stained with an amyloid-specific fluorescent dye before the addition of macrophages, an amyloid-depleting drug, and a test compound, respectively, and the amyloid-depleting drug and the test compound are not labeled, and this approach is preferably used to visualize and determine macrophage-mediated amyloid internalization by detecting intracellular fluorescent signals within phagocytic vesicles. Such fluorescent signals are typically point-type fluorescent signals.

[0418] In one embodiment, each of the amyloid-depleting drug and the test compound is labeled with a fluorescent dye, and the tissue section is not stained before adding the amyloid-depleting drug and the test compound, respectively, to the macrophages, and this approach is preferably used to visualize and determine macrophage-mediated amyloid fragmentation by detecting a sequential separation of fluorescent signals, preferably point-like fluorescent signals, from the fluorescent signals of the amyloid deposits generated by the binding of each of the labeled amyloid-depleting drug and the test compound to the amyloid deposits.

[0419] In principle, any fluorescent dye suitable for amyloid staining—i.e., amyloid-specific fluorescent dyes—or suitable for drug labeling may be used in the method of the present invention. In a preferred embodiment, each of the amyloid-depleting drug and the test compound is labeled with the fluorescent dye Vivotag-680. Additionally or alternatively, the amyloid deposits are preferably stained with Amytracker 680, which is an amyloid-specific dye, or any other Amytracker variant, e.g., Amytracker 480, 520, 540, or 630. Alternatively, Thioflavin T staining may be performed.

[0420] In one embodiment, live cell imaging is performed using a Nanolive CX-A instrument using refractive index imaging for cell visualization and fluorescence microscopy (Cy5 channel) for amyloid imaging. The Nanolive CX-A is preferably placed in a temperature-controlled room and mounted on a vibration-damping table. During imaging, cell culture dishes containing tissue sections were maintained at 37°C under controlled humidity and a 5% CO2 supply. Preferably, imaging occurs every 15 minutes, and the duration of the experiment is preferably about 20 hours.

[0421] Therefore, with the help of high-resolution live cell imaging, not only the binding of amyloid-depleting drugs and test compounds to amyloid deposits, but also macrophage recruitment to amyloid deposits can be observed, and this recruitment leads to the internalization and fragmentation of amyloid and amyloid deposits, respectively, followed by intracellular degradation of amyloid.

[0422] In the method of the present invention, if any one of (a) binding of the amyloid-depleting drug and the test compound to amyloid, (b) amyloid internalization, and (c) amyloid fragmentation is observed, this respectively demonstrates that the amyloid-depleting compound actually possesses amyloid-depleting activity and the test compound possesses amyloid-depleting activity. Preferably, if (b) amyloid internalization or (c) amyloid fragmentation is observed, this respectively indicates that the amyloid-depleting compound actually possesses amyloid-depleting activity and the test compound possesses amyloid-depleting activity. More preferably, if (b) amyloid internalization and (c) amyloid fragmentation are observed, this respectively indicates that the amyloid-depleting compound actually possesses amyloid-depleting activity and the test compound possesses amyloid-depleting activity. More preferably, if (a) binding of the amyloid-depleting drug and the test compound, respectively, to amyloid, (b) amyloid internalization, and (c) amyloid fragmentation are observed, this indicates that the amyloid-depleting compound actually possesses amyloid-depleting activity and that the test compound possesses amyloid-depleting activity, respectively.

[0423] The method of the present invention uses macrophages. Macrophages are immune cells originating from embryonic development or the differentiation of monocytes. Depending on their origin and tissue distribution, and in response to various stimuli and tissue environments, they can adopt numerous phenotypes. Therefore, in vivo, macrophages are rarely strictly classified as pro-inflammatory or anti-inflammatory and possess a continuous phenotype exhibiting a broad expression profile across the entire polarization spectrum. Schematically, the following three major macrophage subpopulations coexist in human tissues: Naive (also called M0) ) Macrophages, pro-inflammatory macrophages referred to as M1 macrophages, and anti-inflammatory macrophages also known as M2 macrophages. Naive macrophages exhibit phagocytic function, recognize pathogenic factors, and rapidly polarize into pro-inflammatory or anti-inflammatory macrophages to acquire a full functional panel. Pro-inflammatory macrophages are extensively involved in inflammatory responses, during which they exert antimicrobial and antitumor functions. In contrast, anti-inflammatory macrophages are involved in the resolution of inflammation, the phagocytosis of cellular debris, and tissue repair after injury. Macrophages also play important roles, either harmful or beneficial, in the initiation and progression of various pathophysiological environments, including solid tumors and hematopoietic cancers. In principle, any type of macrophage can be used as long as it is capable of performing phagocytosis against amyloidogenic proteins.

[0424] As described in Example 5, macrophages were generated by the differentiation of THP1 cells; therefore, in a preferred embodiment, the macrophages used in the method of the present invention are THP1 cell-derived macrophages. In a preferred embodiment, macrophages were generated by the differentiation of THP1 cells with phorbol 12-myristate-13-acetate (PMA)—preferably used at a concentration of 100 ng / mL—and culture was preferably performed for 3 days, followed preferably by a resting period of 3 days without PMA. Macrophages are preferably distributed in tissue sections at a density of 400,000 cells in the presence of an amyloid-depleting drug. If the amyloid-depleting drug to be analyzed is an antibody, preferably an anti-TTR antibody, most preferably an anti-TTR antibody as defined above, the antibody is preferably used at a concentration of 10 nM (1.5 µg / mL). This concentration was previously confirmed to maximally trigger antibody-dependent phagocytosis of ATTR aggregates by macrophages in vitro.

[0425] Amyloid-depleting drugs, also referred to as amyloid-depleting compounds, can remove or reduce amyloid from tissues, preferably through the mobilization of macrophages that internalize and degrade amyloid. In a preferred embodiment, the compound is an anti-amyloid antibody. This mechanism of action is also referred to as antibody-dependent cell-mediated phagocytosis (ADCP). This is defined as a highly regulated process in which an antibody removes a target by linking its Fc domain to a specific receptor on a phagocyte and inducing phagocytosis. In the context of the present invention, ADCP refers to mechanism(s) in which the Fc receptor of a phagocyte (hereafter a macrophage) binds to an amyloid-depleting drug, e.g., an anti-amyloid antibody, which is bound to amyloid, and stimulates the phagocyte to internalize the protein and the cyclic compound, respectively. Thus, the amyloid-depleting drug is preferably an antibody containing an Fc domain, or a fragment or derivative thereof, or an antibody fragment fused to a domain capable of inducing phagocytosis, such as the Fc domain.

[0426] The suitability of the live cell imaging method was verified using tissue sections containing TTR amyloid and an anti-TTR antibody, in particular the anti-TTR antibody ALXN2200 as described above. Accordingly, the amyloid-depleting drug is preferably an anti-TTR antibody capable of depleting amyloid TTR, most preferably an anti-TTR antibody as defined above, wherein TTR is wild-type or mutated TTR, preferably wild-type TTR.

[0427] However, it is reasonable to expect that the method of the present invention can be used not only for the verification, screening, quality control, and characterization of anti-TTR antibodies, but also for the verification, screening, quality control, and characterization of any composition capable of depleting TTR amyloid through the recruitment and phagocytosis of macrophages, respectively. Furthermore, since macrophage-mediated amyloid depletion is not limited to the depletion of TTR amyloid, the above method may be applied to any amyloid-depleting compound, for example, amyloidogenic α-synuclein (α-syn), tau, prion protein (PrP), amyloid beta (Aβ), β2-microglobulin (β2-m), immunoglobulin light chain (LC), immunoglobulin heavy chain (HC), serum amyloid A (SAA), amylin (IAPP), chromosome 9 open reading frame 72 (C9orf72), TAR DNA-binding protein 43 (TDP-43), superoxide dismutase 1 (SOD1), fused RNA-binding protein in sarcoma (FUS), huntingtin (htt), optineurin (OPTN), neuroserpine, ABri, Adan, ubiquiline, optineurin, leukocyte chemotactic factor 2 (LECT2), It may be applied to compounds that deplete gelsoline, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, cystatin C, and lysozyme. Additional amyloid fibrill-forming proteins may be obtained from AmyPro, an open-access database providing a set of amyloid fibrill-forming proteins, and / or (reference [Varadi et al ., Nucleic Acids Research 46 (2018), D387―D392, DOI: 10.1093 / nar / gkx950]), Reference Benson et al. It may be those listed in Table 1 of Amyloid 25 (2018), 215-219.

[0428] In a preferred embodiment, the amyloidogenic protein involved in systemic amyloidosis is selected from the following list: transthyretin (TTR), in particular wild-type TTR and variant TTR, preferably wild-type TTR, immunoglobulin light chain (LC), immunoglobulin heavy chain (LH), serum amyloid A (SAA), leukocyte chemotactic factor 2 (LECT2), gelsoline, apolipoprotein AI (ApoAI), apolipoprotein AII (ApoAII), apolipoprotein AIV (ApoAIV), apolipoprotein CII (ApoCII), apolipoprotein CIII (ApoCIII), fibrinogen, β2 microglobulin, in particular wild-type and variant β2 microglobulin, cystatin C, ABriPP, prion protein, and lysozyme; e.g., the literature [Benson et al. , Amyloid 25 (2018), 215―219] and literature [Muchtar et al. Refer to Journal of Internal Medicine 289 (2021), 268-292.

[0429] The tissue section is preferably a section of heart tissue, kidney tissue, liver tissue, gastrointestinal tissue, skin tissue, muscle tissue, tongue tissue, adipose tissue, salivary gland tissue, lymph node tissue, brain tissue, pancreatic tissue, or any amyloidoma. In a preferred embodiment, the tissue section is a heart tissue section. Most preferably, the tissue section is obtained from a subject suffering from amyloidosis or an amyloid-related disease, preferably from TTR amyloidosis, and most preferably from cardiac TTR amyloidosis. The tissue section obtained from the patient is a tissue section containing amyloid deposits.

[0430] The method of the present invention may also include the analysis of a control compound, which is, for example, an allotypical control antibody, particularly when the amyloid-depleting compound is an antibody. This ensures that the effects observed in the method of the present invention, namely (a) binding of the amyloid-depleting drug and the test compound, respectively, to amyloid, (b) amyloid internalization, and (c) amyloid fragmentation, are attributed to the action of the amyloid-depleting drug and are not due to any non-specific side effects.

[0431] As mentioned above, in a preferred embodiment, an amyloid-depleting drug suitable for TTR amyloid depletion and thus suitable for the treatment of transthyretin (TTR) amyloidosis or TTR amyloid-related diseases, preferably cardiac TTR amyloidosis, is analyzed.

[0432] As shown in Example 5, the mechanism of action of the ATTR-specific antibody was analyzed, and it was found that the antibody binds to amyloid within tissue sections and induces macrophage-mediated amyloid internalization and fragmentation. Accordingly, in one embodiment of the method of the present invention, the drug comprises an anti-amyloid protein antibody or an amyloid-binding molecule. In further embodiments, the drug is an antibody, and the control is a corresponding allotype control antibody. Suitable antibody candidates are known from the prior art, for example, anti-transthyretin (TTR) antibodies are disclosed in WO 2015 / 092077 A1, WO 2014 / 124334 A2, WO 2018 / 007923 A3, WO 2016 / 120810 A1, US 2017 / 0058023 A1 and US 9,879,080 B2. Of course, it is taken into account within the present invention that particularly novel antibodies or compounds may generally be applied to the method of the present invention to evaluate their suitability as amyloid-depleting drugs. In addition to antibodies, any type of drug capable of mobilizing macrophages may be conveniently tested by the method of the present invention for their suitability as anti-amyloid drugs.

[0433] Accordingly, the method of the present invention enables the acquisition and selection of an amyloid-depleting compound, in particular an antibody, which selectively binds to a target amyloid protein at a desired and necessary site of toxic amyloid deposits in a patient to be treated, for example, and can be reasonably expected to be specific enough to mediate the depletion of amyloid.

[0434] In a particularly preferred embodiment, the candidate antibody is a humanized antibody, a human-like antibody or a human antibody—preferably a human-derived antibody, most preferably one isolated from human memory B cells—and a recombinant variant thereof, wherein the recombinant variant typically comprises the variable heavy chain and light chain of the original human-derived antibody and a human constant domain, preferably an IgG1 or IgG4 subtype, which is not necessarily identical to the constant domain of the original human-derived antibody.

[0435] A further aspect of the present invention relates to a process for preparing a pharmaceutical composition comprising an amyloid-depleting drug and a pharmaceutically acceptable carrier. In this process, an amyloid-depleting drug determined to be a suitable amyloid-depleting drug by the method of the present invention as described above is mixed with a pharmaceutically acceptable carrier.

[0436] Pharmaceutically acceptable carriers and routes of administration can be obtained from corresponding literature known to those skilled in the art. The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art; for example, literature [Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. 2 nd Edition by Robinson et al. , Humana Press, Totowa, New Jersey, USA, 2003]; [Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems. 2 ndRefer to [Edition by Taylor and Francis. (2006), ISBN: 0-8493-1630-8]. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to a subject in a suitable dose. Administration of suitable compositions can be carried out by various methods. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods. Aerosol formulations, such as nasal spray formulations, comprise a purified aqueous solution or other solution of an active agent together with a preservative and an isotonic agent. Such formulations are preferably adjusted to a pH and isotonic state suitable for the nasal mucosa. Pharmaceutical compositions for oral administration, such as single-domain antibody molecules (e.g., "Nanobody™"), are also considered in the present invention. Such oral formulations may be in the form of tablets, capsules, powders, liquids, or semi-solids. Tablets may comprise a solid carrier, such as gelatin or an adjuvant. Formulations for rectal or vaginal administration may be provided as suppositories having a suitable carrier; [O'Hagan] et al. , Nature Reviews, Drug Discovery See also 2(9) (2003), 727-735. For additional guidance on formulations suitable for various types of administration, see the literature [ Remington's Pharmaceutical Sciences , Mace Publishing Company, Philadelphia, PA, 17 th It can be found in ed. (1985)] and the corresponding revisions. For a brief review of drug delivery methods, see Langer, ScienceRefer to [249 (1990), 1527-1533]. A preferred carrier according to the present invention is a buffer, an enteric agent and / or a surfactant, most preferably all three of these components.

[0437] In one embodiment of the process of the present invention, a pharmaceutical composition is designed for the treatment of ATTR or ATTR-related diseases. Accordingly, amyloidosis is characterized by ATTR deposits in patients, particularly their respective precursor proteins. Most preferably, the pharmaceutical composition is a pharmaceutical composition comprising an amyloid-depleting drug as defined above, in particular an anti-amyloid antibody, most preferably an anti-TTR antibody, in an aqueous formulation as defined above.

[0438] In another aspect, the present invention relates to a method for characterizing, verifying, developing, and / or quality controlling—which also includes batch control—amyloid-depleting drugs suitable for the treatment of amyloidosis or amyloid-related diseases. For example, the amyloid-depleting drug is characterized by a live cell imaging method, and information regarding the drug applied to the method for verifying the amyloid-depleting drug of the present invention as described above is conveyed to a customer, contracting party, or collaborating partner. Furthermore, the drug determined to be a suitable amyloid-depleting drug may be selected, and optionally, the amyloid-depleting drug, or a pharmaceutical composition comprising the amyloid-depleting drug, is used for the treatment of amyloidosis or amyloid-related diseases.

[0439] The method of the present invention may be carried out in addition to other methods useful for verifying the therapeutic applicability of a candidate drug and / or for detecting the ability of a drug to deplete amyloidogenic proteins and amyloid deposits, respectively. Such a method is a method using a non-human animal model of a patient-derived amyloid xenograft (PDAX) as disclosed, for example, in WO 2020 / 094883 A1, the contents of which are incorporated herein by reference.

[0440] Examples

[0441] The following specific embodiments for carrying out the present disclosure are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0442] Example 1. Cloning and expression of NI006 / ALX2220

[0443] NI006 / ALXN2220 is produced in the CHO K1 cell line (ATCC No. CCL 61).

[0444] Construction and sequence verification of the expression plasmid

[0445] First, a signal peptide was added to the sequences encoding the heavy chain gene (presented in sequence number 15) and the light chain gene (presented in sequence number 16) of NI006 / ALXN2220, and each expression cassette (heavy chain gene containing the signal peptide and light chain containing the signal peptide) was cloned into individual expression vectors.

[0446] The expression vector contains typical elements such as a promoter for controlling gene expression, a ribosome entry site, two selectable markers—one for maintenance in mammalian cells and one for maintenance in E. coli—a terminator, and a replication origin for proliferation in E. coli.

[0447] Before use for transfection, the CHO-K1 host cell line was thawed and cultured. Linearized light and heavy chain expression vectors were transfected into the CHO-K1 host cell line in a 1:1 ratio using Freestyle Max reagents. At 48 hours after transfection, cells were plated in four 96-well plates in a selective medium containing the corresponding antibiotic for maintenance in mammalian cells. The 96-well plates were incubated in a static CO2 incubator (36.5°C, 6% CO2) for 2 to 3 weeks, with the medium replaced every 3 to 4 days.

[0448] Subsequently, a single clone was tested, and a master cell bank (MCB) was manufactured.

[0449] Example 2. Characterization of mature NI006 / ALXN220

[0450] The antibody NI006 / ALXN2220 was generated in the CHO-K1 cell line (ATCC No. CCL 61) as described in Example 1 and obtained from the cell culture medium. The amino acid sequences of the mature heavy chain (HC) and light chain (LC) of NI006 / ALXN2220 are presented in Sequence Nos. 7 and 8 with the modifications mentioned below. The total number of amino acids, the number of amino acids in the heavy chain, and the number of amino acids in the light chain are 1328, 450, and 214, respectively.

[0451] Further characterization of the antibody NI006 / ALXN2022 was performed primarily by standard procedures, e.g., mass spectrometry analysis. For example, post-translational modifications of NI006 / ALXN2220 were identified using tandem mass spectrometry (LC-MS / MS) analysis and free sulfhydryl analysis of fragments of NI006 / ALXN2220 obtained from Lys-C and trypsin sequential digestion. Characterization of antibody-based therapeutics via LC-MS analysis is a standard procedure and can be performed by those skilled in the art; see, for example, the literature [Robotham and Kelly, Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics (2020), 1-33].

[0452] Exemplary method for freed N-glycan for determination of glycosylation profile

[0453] The N-glycans of ALXN2220 are profiled using the liberated N-glycan method. The N-glycans of ALXN2220 are liberated using PNGase F and labeled with 2-AB, after which HILIC (Hydrophilic Interaction Chromatography) separation and fluorescence detection (FLD) are performed using an UPLC system. Individual N-glycans are quantified as the percentage of their peak area relative to the total peak area.

[0454] Exemplary Peptide Mapping Study Using LCMS:

[0455] Analysis of primary structure and post-translational modifications is performed using peptide mapping with high-resolution mass spectrometry (MS). ALXN2220 is reduced, denatured, and alkylated. The reduced and alkylated proteins undergo sequential degradation by endoproteinase Lys-C / trypsin. The resulting degraded peptides are separated using a reverse-phase C18 column and detected by a high-resolution mass spectrometer. Peptide identification is confirmed by the theoretical mass of the peptide and MS / MS. Post-translational modifications are quantified as the percentage of the peak area of ​​the modified peptide relative to the sum of the peak areas of the modified and unmodified forms of the peptide.

[0456] Exemplary icIEF method (charge method) for determining charge variants:

[0457] Imaging Capillary Isoelectric Focusing (iCIEF) is used to separate charge variants of proteins based on their isoelectric points (pI). Samples are prepared by mixing with a master mixture consisting of Pharmalyte, a pI marker, urea, and water. ALXN2220 is separated into main peaks, acidic regions, and basic regions via a pH gradient formed by a carrier amphoteric substance inside the capillary, and isoforms are separated according to their isoelectric points. The focused protein regions are detected by whole-capillary UV detection. Acidic, main, and basic species are quantified as the percentage of their peak area relative to the total of all detected protein peaks.

[0458] The molecular weight of antibody NI006 / ALXN2220, measured by standard mass spectrometry, is approximately 147.1 kDa for intact IgG1 and 144.2 kDa for the deglycosylated variant. The pIs of antibody NI006 / ALXN2220 are 8.4 (theoretical value) and 9.3 (experimentally determined value). The theoretical quenching coefficient (A) of antibody NI006 / ALXN2220 0.1% )(mL / (mg * cm)) is 1.390 (theoretical value) and 1.438 (experimentally determined value).

[0459] The monoclonal antibody NI006 / ALXN2220 is an IgG1 subclass antibody composed of two heavy chains of the IgG1 subclass and two light chains of the kappa subclass. The four chains are stabilized by multiple disulfide bonds. In particular, as determined by standard procedures, namely Lys-C and trypsin digestion and subsequent LC-MS, at least the following disulfide crosslinks are present in NI006 / ALXN2220:

[0460] LC:C23-LC:C88;

[0461] LC:C134-LC:C194;

[0462] LC:C214-HC:C223;

[0463] HC:C22-HC:C97;

[0464] HC:C147-HC:C203;

[0465] HC1:229-HC2:229 and HC1:232-HC2:232;

[0466] HC:C264-HC:C324;

[0467] HC:C370-HC:C428;

[0468] NI006 / ALXN2220 is a glycoprotein, and the constant region of each heavy chain contains a single N-linked glycan site at residue N300. During the determination of the glycosylation profile, the major N-glycan types were found to be G0F (approx. 49.0%) and G1F (approx. 25.4%). More specifically, the following glycosylation profile (sugar types, positions of glycosylation sites, etc.) was determined for NI006 / ALXN2220:

[0469]

[0470] The nomenclature of glycans follows the order HexNac-Hexos-Fucose-NeuAc-NeuGc. For example, 23000 is HexNac(2)-Hexos(3)-Fucose(0)-NeuAc(0)-NeuGc(0). G1Fa and G1Fb are isomers and are grouped as G1F. G1F is calculated as the sum of G1Fa and G1Fb using the original unrounded numbers.

[0471] The glycosylation profile is shown in Figure 6, and the abundance of different glycans in 11 individual batches of NI006 / ALXN2220 is listed in Table 2.

[0472] [Table 2]

[0473]

[0474] Furthermore, N-terminal glutamine modified as heavy-chain pyroglutamic acid (sample abundance: 99.9%) and C-terminal lysine clipping (sample abundance: 95.8%) was identified as major post-translational modifications. Additionally, modifications of a small proportion, such as methionine oxidation, asparagine deamidation, and asparagine succinimide formation, were experimentally determined (e.g., using LCMS-based methods) and further predicted as presented in Tables 3 and 4:

[0475] [Table 3]

[0476]

[0477] [Table 4]

[0478]

[0479] The abundance of post-translational modifications of antibody NI006 / ALXN2220 in samples is presented in Table 5:

[0480] [Table 5]

[0481]

[0482] In summary, N-linked glycosylation of the heavy chain, N-terminal pyro-glutamic acid modified from N-terminal glutamine, and C-terminal lysine clipping of the heavy chain are the major post-translational modifications of NI006 / ALXN2220.

[0483] Furthermore, charge variant analysis was performed, and the results are listed in Table 6.

[0484] [Table 6]

[0485]

[0486] Example 3. ALXN2220 Manufacturing Process

[0487] In this example, the scaling up of the ALXN2220 drug substance (DS) manufacturing process from a 1-phase 500 L scale (Process A1) to a 3-phase 2000 L scale (Process A2) is described, and major upstream process changes are summarized in Table 7.

[0488] The product quality results of ALXN2220 at the time of DS shipment are summarized in Table 7. In particular, the imaged capillary isoelectric focusing (iCIEF) acidity % of the first 2000 L DS batch (GMP 1(A2)) was significantly higher than the previous results from process A1 at the 500 L scale (Fig. 1).

[0489] To mitigate the risk of high acidity observed in the first 2000 L DS batch (GMP 1(A2)), a number of upstream process parameters were evaluated using a small-scale bioreactor, and among these, the most effective parameter for reducing acidity was identified as the effective pH controlled by the pH set point and dead band. When a pH dead band of 0.05 was applied (culture ID: #1), the effective pH (reflected as online pH) was approximately 0.05 and 0.15 lower than the pH dead bands of 0.10 (culture ID: #2) and 0.20 (culture ID: #3), respectively, for most of the cell culture generation stage (days 5 to 14) (Fig. 5).

[0490] [Table 7]

[0491]

[0492] After purifying cell culture samples from all three bioreactors with small-scale protein A, product quality tests were conducted (Tables 8 and 9). Small-scale data showed that reducing the pH dead band from 0.20 to 0.05 resulted in a significant reduction in acidic species (Fig. 3). Surprisingly, reducing the pH dead band from 0.20 to 0.05 also led to a reduction in Man5 (Fig. 4)—showing more robust control over gomannose species—with no significant adverse effects on other tested product quality attributes (Tables 8 and 9).

[0493] After small-scale experiments confirming the effectiveness of the pH control strategy (mentioned above) on acidic species and Man5 reduction, two additional upstream parameters (target seeding density and temperature-switched viable cell density range) were further modified to verify process robustness before implementation in a 2000 L-scale process A2 starting from a second 2000 L batch. The DS release results from iCIEF (Fig. 5) and N-glycan (Fig. 2) at the 2000 L scale confirmed the effectiveness and robustness of the iCIEF acidity and Man5 control strategy for ALXN2220. Glycosylation profiles were determined using the liberated N-glycan method mentioned above.

[0494] [Table 8]

[0495]

[0496] [Table 9]

[0497]

[0498] [Table 10]

[0499]

[0500] In summary, reducing the pH dead band from 0.20 to 0.05 resulted in a surprising decrease in Man5—as can be seen in Figure 4 as well as Table 9—which clearly shows that batch GMP1(A2)(pH dead band: 0.2) has a significantly higher Man5 content than all other batches GMP2(A2) to GMP9(A2)(pH dead band: 0.05).

[0501] Example 4. Stability study of anti-TTR monoclonal antibody formulation

[0502] The formulation development of NI006 / ALXN2220 included studies designed to select buffer systems and excipients to stabilize the protein. The formulation was developed to prevent product loss and minimize the reduction in purity and bioactivity due to stresses faced during production, storage, transport, and handling.

[0503] A pH buffer screening study was conducted to determine the optimal buffer system for the drug product formulation. A 20 mM histidine buffer with a pH of 5.8 was selected as the final buffer system.

[0504] Different types of excipients, including disaccharides (e.g., sucrose and trehalose), amino acids (e.g., L-arginine HCl), polyhydric alcohols (e.g., sorbitol), and surfactants (e.g., polysorbate 80), were evaluated through excipient studies. 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 optimal excipients for the NI006 / ALXN2220 formulation because they were shown to maintain product purity by minimizing the reduction in SEC, cIEF, and caliper purity.

[0505] In the excipient concentration screening study, three different polysorbate 80 concentrations (0.02%, 0.04%, and 0.06% (w / v)) and two sucrose concentrations (6.5% and 8% (w / v)) were finally tested. Three stress conditions were used for screening: stirring, heat, and freeze-thaw. In the stirring study, samples were placed at 25°C with stirring at 200 rpm for up to 7 days or without stirring. In the heat study, samples were placed at 40°C for up to 2 weeks. And in the freeze-thaw study, samples were frozen and thawed for up to 5 cycles. Appearance, pH, protein concentration, number of invisible particles, and purity were evaluated.

[0506] A polysorbate 80 concentration of 0.03% (w / v) was selected as the surfactant strength because the formation of invisible particles was effectively suppressed 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 for 1 month at 40°C or 3 months at 25°C, no substantial differences were observed between the 6.5% and 8% (w / v) sucrose formulations in terms of DSC (differential scanning calorimetry), appearance, pH, protein concentration, SEC, cIEF, CE-SDS (non-reducing and reducing), invisible particles, and efficacy.

[0507] The final formulation developed is NI006 / ALXN2220 at a target concentration of 50 mg / mL and a pH of 5.8 in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80. Excipients were selected based on their stabilizing effects on the drug product. L-histidine and L-histidine monohydrochloride stabilize the pH in the liquid state at a concentration of 20 mM. Sucrose modifies the osmolal concentration to isotonic at a concentration of 8% (w / v), stabilizes the NI006 / ALXN2220 protein against aggregate formation in the liquid state, and acts as a cryoprotectant during freezing and thawing. Polysorbate 80 at a concentration of 0.03% (w / v) was selected to stabilize the NI006 / ALXN2220 protein against surface-induced protein denaturation or aggregation in the liquid state.

[0508] Manufacturing process development

[0509] The drug product manufacturing process consists of thawing the drug material, pooling and mixing, sterile filtration, aseptic filling, capping, visual inspection, and bulk packaging. Sterile filtration was selected as a method to obtain a sterile drug product and was performed using two serially connected sterile filters (0.22 μm, PVDF). Before and after sterile filtration, a water bubble point test is performed on the filters to ensure their integrity. Compatibility between the drug product and contact components on the filling line, the influence of shear stress caused by the peristaltic pump, and stability under light exposure were evaluated to mitigate potential adverse effects on product quality attributes during manufacturing. A non-clinical lot (Lot 201901004) and three clinical lots (Lots 201903038, 201904050, and 20200801) were filled. The non-clinical and clinical lots used the same filling volume, container closure system, unit operation sequence, and storage conditions. There were no significant changes in the drug product manufacturing process between the non-clinical and clinical lots. Minor differences are listed below:

[0510] A 2 L scale was used for the non-clinical lot, and a 14 L scale was used for the clinical lot.

[0511] The formulation used in nonclinical lot 201901004 was 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) at pH 5.8. The formulations used in clinical lots 201903038 and 201904050 were 50 mg / mL in 20 mM histidine buffer, 6.5% sucrose (w / v), and 0.03% polysorbate 80 (w / v) at pH 5.8. The formulation used in clinical lot 20200801 was 50 mg / mL in 20 mM histidine buffer, 8% sucrose (w / v), and 0.03% polysorbate 80 (w / v) at pH 5.8.

[0512] A 5 L glass bottle was used for pooling and mixing drug components for the non-clinical lot, and a 50 L mixing bag was used for pooling and mixing drug components for the clinical lot.

[0513] Thawing, pooling, and mixing of bulk drug components

[0514] Frozen drug components stored in a 2 L PETG bottle are thawed at room temperature (18°C to 24°C) in a light-protected room. After complete thawing, the drug components are pooled into a 50 L mixing bag and stirred at an appropriate speed to observe behavior without generating foam. The mixing time is controlled to 15 to 20 minutes. Before sterile filtration, samples are taken for pH, protein concentration, osmolal concentration, and bioburden.

[0515] Sterilization filtration

[0516] Bulk drug components are sterile filtered in a Grade A environment using a peristaltic pump through two series connected to 0.22 μm sterile filters in 20 L sterile disposable bags. Before and after sterile filtration, filter integrity tests are performed on both filters.

[0517] Aseptic filling

[0518] Aseptic filling is performed inside a RABS unit that completely surrounds the filling material and provides a Class A environment. The RABS unit isolates the operator from the aseptic interior. All filling components are autoclaved and aseptically assembled. Sterile, dethermally sterilized 2 mL (2R) glass vials are filled to a target volume of 2.25 mL. To ensure the filling weight is between 2.233 and 2.442 g / vial, the filling weight is checked periodically during the filling process.

[0519] Putting on a stopper

[0520] The filled vial is automatically capped with a 13 mm rubber stopper inside the RABS unit. The stopper is steam sterilized at 122°C for 30 minutes.

[0521] Capping

[0522] The stoppered vial is transferred via a conveyor belt to a capping machine under Class A laminar flow protection. The stoppered vial is capped with a 13 mm plastic aluminum flip-off cap. The cap is steam sterilized at 122°C for 30 minutes.

[0523] Visual inspection

[0524] Manual 100% visual inspection is performed on capped vials by production staff, followed by statistically based AQL (Acceptable Quality Limit) inspection by quality assurance. Release and stability samples are taken after the visual inspection.

[0525] Bulk packaging and storage

[0526] After that, the filled drug product vials are bulk packaged and labeled. The bulk-packaged drug product vials are stored at 2°C to 8°C.

[0527] Container closure system

[0528] 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 flip-off aluminum cap. The components were selected for their durability against sterilization and depyrogenation processes, as well as for non-reactive contact surfaces that are optimally compatible with proteins. The compatibility of the container closure system and the drug product is evaluated by accelerated and long-term stability studies presented below.

[0529] The integrity of the container closure system was verified by a dye penetration test. Drug product vials were immersed in a colored dye, maintained under vacuum, then depressurized, and penetration into the vials was examined. The container and closure system achieved 100% airtightness. Container closure integrity testing (CCIT) is performed annually within the stability program using a non-destructive vacuum depressurization method. Product contact materials, glass vials, and rubber stoppers were tested in accordance with U.S. and European Pharmacopoeia requirements and are suitable for parenteral use. Regulatory compliance is verified based on the Certificate of Conformity provided by the supplier for each vial and stopper.

[0530] Analysis Procedure

[0531] color

[0532] Color conforms to European Pharmacopoeia 2.2.2. The chromatic aberration method is used for color measurement.

[0533] transparency

[0534] Transparency complies with European Pharmacopoeia 2.2.1. The light scattering method is used to measure transparency.

[0535] pH

[0536] United States Pharmacopoeia <791> and complies with European Pharmacopoeia 2.2.3. The potentiometric determination method is used for pH measurement.

[0537] Osmolal concentration

[0538] United States Pharmacopoeia <785> and comply with European Pharmacopoeia 2.2.35. Osmolal concentration is determined indirectly by measuring the freezing point depression of the solution.

[0539] iCIEF

[0540] Whole Column Imaging 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 charged variant species within a protein sample. pI is an intrinsic characteristic of a specific protein molecule and is the pH at which the protein molecule carries no net charge. Under an external electric field, charged variants move along a continuous pH gradient formed by an amphoteric electrolyte and stop where the pH is equal to their pI. At that pI, the protein carries no net charge and is not attracted by either of the electrodes. Consequently, different monoclonal antibody species with different pI values ​​are separated and focused at different locations. The pI values ​​and relative abundance of the separated peaks can be identified and quantified using chromatography software.

[0541] To satisfy the acceptance criterion of "conforms to the profile of the reference standard," the electrophoretic graph must show a peak profile comparable to that of the reference standard. In addition, the difference in the pI value of the main peak between the tested sample and the reference standard (average) must be 0.2 or less.

[0542] SEC-HPLC

[0543] Size exclusion chromatography-high-performance liquid chromatography (SEC-HPLC) is a purity analysis method that separates proteins based on their size. The stationary phase consists of inert particles packed within a dense three-dimensional matrix. The particles have small pores that allow only species smaller than a specific size to enter. Larger molecules are too large to enter the pores and simply pass by them. Consequently, larger molecules flow through the column faster than smaller molecules; the smaller the molecule, the longer the retention time. After separation, the relative percentages of high molecular weight (HMW) species, monomers, and low molecular weight (LMW) species are quantified via UV detection.

[0544] CE-SDS (reduction)

[0545] Reduction capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is a purity analysis method that separates proteins based on their electrophoretic mobility, where smaller proteins migrate faster than larger proteins. In this method, the test sample is denatured by heating in the presence of SDS. The sample is reduced by adding the reducing agent beta-mercaptoethanol (BME) to the sample solution. Separation is performed through an uncoated capillary, and the protein sample is detected at 220 nm using a photodiode array (PDA) detector. The results are reported as a purity percentage.

[0546] CE-SDS (non-reducing)

[0547] CE-SDS (non-reducing) is a purity analysis method that separates proteins based on their electrophoretic mobility, where smaller proteins migrate faster than larger 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 sulfhydryls from binding to other sulfhydryls. Separation is performed 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.

[0548] biological load

[0549] The biological load test in the United States Pharmacopoeia (USP) <61> and is performed by membrane filtration based on European Pharmacopoeia (Ph. Eur.) 2.6.12. 10 mL of the drug component sample is filtered through the sterile surface of a 0.45 μm membrane. Afterward, the filter membrane is transferred onto a culture plate of soy-casein hydrolyzed agar for the determination of the total aerobic microbial count (TAMC). Another filter membrane used for filtering the 10 mL drug component sample is transferred onto a culture plate of sabrowd dextrose agar for the determination of the total combined yeasts and molds count (TYMC).

[0550] Endotoxin

[0551] Bacterial endotoxin testing is performed by kinetic turbidity assay, and the U.S. Pharmacopoeia <85> It is established based on the European Pharmacopoeia 2.6.14. Endotoxins produced by Gram-negative bacteria are detected using horseshoe crab amebocyte lysates that coagulate with the endotoxin. By establishing a 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.

[0552] ELISA (linking assay)

[0553] The binding efficacy of the NI006 / ALXN2220 antibody is evaluated using the ELISA method. Samples, controls, and reference standards in appropriate dilutions are loaded onto misfolded TTR (antigen for NI006 / ALXN2220) coated 96-well plates. After washing the plates, horseradish peroxidase (HRP)-conjugated goat anti-human IgG is added to the wells to enable interaction with the bound NI006 / ALXN2220 antibody captured by the misfolded TTR. After the final washing step, TMB substrate solution is loaded into the wells. TMB specifically reacts with peroxide in the presence of peroxidase and generates a colorimetric signal proportional to the amount of NI006 / ALXN2220 protein bound to the wells. Color development is stopped, and the optical density is measured at 450 nm (minus 560 nm for wavelength calibration). Plot the dose-response curves of the sample and reference standard according to a 4-parameter logistic (auto-estimated) regression model using SoftMax Pro GxP software. Calculate individual EC50 values ​​for the sample and reference standard. Calculate the relative binding activity of the sample using the following formula:

[0554] Relative binding activity of sample (%) = (EC50 of reference standard / EC50 of sample) × 100%

[0555] Protein concentration

[0556] Proteins in solution absorb ultraviolet light at a wavelength of 280 nm due to the presence of aromatic amino acids within the protein molecules. 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 cellular path length (l), and the protein's extinction coefficient (ε) as follows: A = C | ε. Unlike traditional UV-Vis methods that rely on a single absolute absorbance value, gradient spectroscopy uses section data (absorbance versus path length) to determine the gradient value for the quantification of sample concentration using the gradient spectroscopy equation (gradient = ε C) derived from the Beer-Lambert law.

[0557] Cell-based assay

[0558] THP-1 is a human monocyte 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 to mis-TTR in cell culture medium. Briefly, THP-1 cells at a rate of approximately 2 × 10⁴ cells per well in assay medium were seeded into a 96-well cell culture plate at a rate of 100 μL / well, and then a mixture of serial dilutions of NI006 / ALXN2220 mAb standard (final concentration: 2000 to 0.039 g / mL) and misfolded-TTR (final concentration: 10 μg / mL) was loaded into duplicate wells at a rate of 100 μL / well. After incubating with 5% CO2 at 37°C for 20 to 24 hours, IL-8 production is measured using a human IL-8 ELISA kit.

[0559] Commercialization Research

[0560] The compatibility of NI006 / ALXN2220 with substances in clinical use was evaluated. Compatibility was evaluated with the following clinical administration settings:

[0561] Bags and injection lines under PVC setup - IV bags, IV lines, and filters are made of PVC material.

[0562] Bags and injection lines under a non-PVC setup - IV bags, IV lines, and filters are made of non-PVC material.

[0563] Syringe under PVC setting - Perfusion line and filter are made of PVC material

[0564] Syringe under non-PVC setup - perfusion line and filter are made of non-PVC material

[0565] Three concentrations (1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL) were tested for 24 hours at 2°C to 8°C, followed by testing at 25°C for 6 hours (total 30 hours). Saline solution and glucose were used as diluents for the 1.0 mg / mL and 20.0 mg / mL concentrations. The results are presented in Tables 11 and 12.

[0566] [Table 11]

[0567]

[0568] [Table 12]

[0569]

[0570] In the saline group, visible particles were observed, indicating that NI006 / ALXN2220 is less stable in saline. The data show that when glucose was used as a diluent, no substantial changes were observed in appearance, protein concentration, invisible particles, SEC-HPLC, and ELISA binding assays. At concentrations of 1.0 mg / mL, 20.0 mg / mL, and 50.0 mg / mL, NI006 / ALXN2220 is stable for 24 hours at 2°C to 8°C, followed by 6 hours at 25°C (total 30 hours). NI006 / ALXN2220 is commercially available with the evaluated clinical use material.

[0571] A concentration of 0.15 mg / mL, which is 10 times lower than the lowest dose concentration in clinical trials, was also investigated. Variations were observed in protein concentration and in ELISA tests. Variations in ELISA tests may be caused by fluctuations in protein concentration resulting from protein adsorption to the contact material. In conclusion, it was decided to use 5% glucose as a diluent for clinical use.

[0572] In Table 13 below, batch analysis data for nonclinical lot 201901004 and clinical lot 201903038 are listed as examples.

[0573] [Table 13]

[0574]

[0575] As a reference standard, a composition containing 50 mg / mL of antibody formulated at pH 5.8 in 20 mM histidine buffer, 8% (w / v) sucrose, and 0.03% (w / v) PS80, stored at -70 ± 10°C in a vial filled with 100 μl was used. Additional tests were performed to quantify the reference standard, and the results are presented in Table 14.

[0576] [Table 14]

[0577]

[0578] Protein concentration and efficacy were corrected for the reference standard. The efficacy of the reference standard is specified as a value of 100% relative efficacy.

[0579] Stability study

[0580] Non-clinical lot 201901004 and clinical lot 201903038 were placed in stability tests. Non-clinical lot (201901004) has stress stability data for 1 month, accelerated stability data for 6 months, and long-term stability data for 18 months. Clinical lot (201903038) has stress stability data for 1 month, accelerated stability data for 6 months, and long-term stability data for 12 months. Under stress conditions, both clinical lot (201903038) and non-clinical lot (201901004) showed a trend of significant decrease in the iCIEF main peak (%) and increase in the acid peak (%), while no significant changes were observed in other purity tests as well as in the ELISA binding test.

[0581] The shelf life for the drug product is currently set to 24 months when stored at 5°C to 3°C in a light-protected environment. The available stability and compatibility data presented above indicate that the infusion solution is stable for up to 24 hours at 2°C to 8°C and subsequently stable for 6 hours at 25°C after dilution with a 5% glucose solution. From a microbiological perspective, the infusion solution must be used immediately. If not used immediately, the storage period in use is set to 4 hours at room temperature or 24 hours at 2°C to 8°C. Tables 15 through 18 summarize the available stress data for nonclinical lot 201901004 and clinical lot 201903038.

[0582] [Table 15]

[0583]

[0584] [Table 16]

[0585]

[0586] [Table 17]

[0587]

[0588] [Table 18]

[0589]

[0590] Tables 19 through 22 summarize available accelerated data for nonclinical lot 201901004 and clinical lot 201903038.

[0591] [Table 19]

[0592]

[0593] [Table 20]

[0594]

[0595] [Table 21]

[0596]

[0597] [Table 22]

[0598]

[0599] [Table 23]

[0600]

[0601] [Table 24]

[0602]

[0603] [Table 25]

[0604]

[0605] [Table 26]

[0606]

[0607] [Table 27]

[0608]

[0609] [Table 28]

[0610]

[0611] As can be seen from the stress stability, accelerated stability, and long-term stability data presented in Tables 11 through 28, the tested pharmaceutical formulations are stable over the long term. For example, the formulations remain liquid without any visible particles, the pH remains constant, and the monomer content does not drop below 96% as measured by SEC-HPLC, meaning that the content of HMWS and LMWS remains below 4% under all tested conditions. Furthermore, the amount of acidic species does not exceed 40% during the long-term stability test as measured by iCIEF, and the ELISA binding assay demonstrated that the antibody maintains its binding ability (it did not drop below 70% of the reference standard under all tested conditions, and did not even drop below 95% during the long-term stability test).

[0612] Example 5: As demonstrated using high-resolution live cell imaging, the antibody NI006 / ALXN2220 mediates amyloid depletion from patient heart tissue.

[0613] Extracellular deposition of transthyretin amyloid (ATTR) is a characteristic of ATTR cardiomyopathy, and reducing cardiac ATTR load is a key therapeutic goal for improving cardiac function. In this study, the cellular mechanisms of ALXN2220-mediated ATTR depletion in patient-derived cardiac tissue were visualized using high-resolution live-cell imaging.

[0614] method

[0615] Frozen myocardial tissue samples containing ATTR amyloid deposits were prepared into sections with a diameter of 8 mm and a thickness of 15 μm using a cryostat. The sections were mounted on a 35 mm micro dish specialized for imaging with the Nanolive system and stored at -20°C until use.

[0616] Two methods were used to visualize amyloid deposits within tissue sections. One approach involved staining amyloid deposits with the amyloid-specific dye Amytracker 680 (Ebba Biotech (1 mg / mL in H2O)). A 1 µg / mL working solution of Amytracker 680 was prepared in H2O and applied to tissue sections at room temperature for 5 minutes. After destaining with 70% alcohol and washing with H2O, the amyloid depletion assay was performed. The other approach involved using the antibody NI006 / ALXN2220, labeled with the fluorescent dye Vivotag-680. The antibody labeling reaction was induced for free amines and performed as directed in the instructions; subsequently, dialysis was performed to remove unbound dye.

[0617] THP1 cells (THP1 Null2 cells: Invivogen, reference #thp-nullz, lot: T49-4101) were differentiated for 3 days using 100 ng / mL of phorbol 12-myristate-13-acetate (PMA, Sigma P8139), followed by resting for 3 days without PMA to generate macrophages. Macrophages were detached by trypsin treatment and dispensed into tissue sections at a density of 400,000 per well (micro-dish: reference #80136, μ-dish 35 mm, shallow type (low), polymer coverslip) in the presence of 10 nM (1.5 μg / mL) of NI006 / ALXN2220 or an allotypical control. This concentration was previously confirmed to maximally trigger antibody-dependent phagocytosis of ATTR aggregates by macrophages in vitro.

[0618] Live cell imaging was performed using the Nanolive CX-A instrument with refractive index imaging for cell visualization and fluorescence microscopy (Cy5 channel) for amyloid imaging. The Nanolive CX-A was placed in a temperature-controlled room and mounted on a vibration-damping table. During imaging, the cell culture dishes were maintained at 37°C under controlled humidity and a 5% CO2 supply. For most experiments, imaging occurred every 15 minutes, and the experiment duration was approximately 20 hours.

[0619] To elucidate the mechanism of cardiac amyloid depletion in the presence of NI006 / ALXN220 and macrophages, representative cells exhibiting tissue amyloid phagocytosis were selected. The field of view was focused on the cells and viewpoints of interest, and refractive index imaging and contrast settings for amyloid fluorescence were adjusted for internalized amyloid material.

[0620] result

[0621] Initially, ATTR deposits in myocardial tissue sections were identified using Amytracker 680, a red fluorescent dye specific to amyloid. NI006 / ALXN2220 was labeled with the green fluorescent dye A488, and the selective binding of NI006 / ALXN2220 to ATTR was indicated by the overlap between red and green fluorescence, as shown in Figures 7 (A) to (D).

[0622] It was further revealed that NI006 / ALXN2220 triggered amyloid phagocytosis by macrophages. Dot-like and intracellular red fluorescence patterns, as visualized in Figure 8, indicated the presence of ATTR amyloid within phagocytic vesicles. Macrophages exhibited extensive phagocytic activity, as indicated by the number of red fluorescence vesicles.

[0623] Furthermore, experimental results showed that two-nucleated macrophages were spontaneously formed under culture conditions. Figure 9 shows one such multinucleated cell continuously sloughing off amyloid fragments from a large deposit. The macrophage shown in Figure 10 was observed separating a thin, elongated ATTR deposit from an adjacent cardiomyocyte, moving the deposit more than 20 μm away. The macrophage also cleaved the protruding large amyloid deposit, sloughing off small and large fragments of amyloid.

[0624] In summary, the binding of NI006 / ALXN2220 to ATTR amyloid in patient myocardial tissue sections was demonstrated using fluorescently labeled NI006 / ALXN2220. Furthermore, NI006 / ALXN2220 triggered the macrophage recruitment process toward amyloid deposits, followed by amyloid fragmentation, internalization, and intracellular degradation, whereas multinucleated cells were found to participate in the phagocytosis of large amyloid fragments. In the presence of an allotypical control antibody, macrophages did not phagocytize cardiac ATTR amyloid.

[0625] Therefore, by developing this innovative high-resolution live cell imaging method, we were able to demonstrate NI006 / ALXN2220-mediated amyloid depletion in patient cardiac tissue, which further improved the understanding of the cellular mechanisms underlying antibody-mediated amyloid depletion as a precision therapy for patients with ATTR cardiomyopathy.

Claims

Claim 1 A composition comprising one or more antibodies, wherein the antibody comprises an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (wherein, in one or more of the above antibodies, glutamine (Q) at position 1 is modified to pyroglutamate (pE), lysine (K) at position 450 is absent, and asparagine (N) at position 300 is glycosylated), where Sequence No. 8 is the following sequence Including:DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, where the antibody is of the following glycan type: Man3+1F,A composition comprising G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2. Claim 2 A composition comprising one or more antibodies, wherein the antibody comprises an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (wherein, in one or more of the above antibodies, glutamine (Q) at position 1 is modified to pyroglutamate (pE), lysine (K) at position 450 is absent, and asparagine (N) at position 300 is glycosylated), where Sequence No. 8 is the following sequence A composition comprising: DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, wherein less than 3% of the glycan is of the Man5 type. Claim 3 A composition comprising one or more antibodies, wherein the antibody comprises an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (wherein, in one or more of the above antibodies, glutamine (Q) at position 1 is modified to pyroglutamate (pE), lysine (K) at position 450 is absent, and asparagine (N) at position 300 is glycosylated), where Sequence No. 8 is the following sequence Including:DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, where the antibody is of the following glycan type: Man3+1F,A composition comprising G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, wherein less than 3% of the glycan is of the Man5 type. Claim 4 A composition according to any one of claims 1 to 3, wherein the major glycan types are G0F and G1F. Claim 5 A composition according to claim 4, wherein approximately 49% of the glycans are of the G0F type and approximately 25% of the glycans are of the G1F type. Claim 6 A composition according to any one of claims 1 to 5, comprising the following charge variants of the antibody: main peak ≥ 50.0%, acidic peak ≤ 40.0%, basic peak ≤ 15.0%. Claim 7 A composition according to any one of claims 1 to 6, comprising the following charge variants of the antibody: main peak ≥ 63.0%, acidic peak ≤ 32.0%, basic peak ≤ 5.0%. Claim 8 A composition according to any one of claims 1 to 7, wherein the antibody is contained in an aqueous formulation, the formulation comprises an antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v, the formulation having a pH of about 5.

8. Claim 9 A composition according to any one of claims 1 to 8, wherein the formulation has a content of acidic species of the antibody ≤ 40.0% and a content of basic species of the antibody ≤ 15.0% after storage at 5 ± 3℃ for 6 months as determined by iCIEF; or a content of high molecular weight species ≤ 5.0% after storage at 5 ± 3℃ for 6 months as determined by SEC-HPLC. Claim 10 A composition according to any one of claims 1 to 9, wherein the antibody is obtainable by a method comprising the steps of: culturing CHO-K1 cells comprising one or more polynucleotides encoding an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, or culturing one or more vectors comprising polynucleotides(s) in Chinese hamster ovary (CHO)-K1 cells, and isolating the antibody from the cell culture medium. Claim 11 The composition of claim 10, wherein the method comprises the step of culturing CHO-K1 cells to a pH dead band of less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ 0.075, preferably ≤ 0.05, more preferably 0.05 or 0.1, and most preferably 0.

05. Claim 12 In any one of claims 1 to 11, in one or more of the antibodies, the asparagine (N) at position 58 is deamidated and / or; the methionine (M) at position 71 is oxidized and / or; the methionine (M) at position 115 is oxidized and / or; the methionine (M) at position 255 is oxidized and / or; the aspartate (D) at position 283 is iso-aspartate and / or; the asparagine (N) at position 318 is deamidated or contains succinimide and / or; the methionine (M) at position 361 is oxidized and / or; the asparagine (N) at position 387 is deamidated or contains succinimide and / or; the methionine (M) at position 431 is oxidized and / or; the proline (P) at position 448 is amidated and / or; and the glycine (G) at position 449 is Absent composition. Claim 13 A composition according to any one of claims 1 to 12, wherein, in more than 90% of the antibody, preferably about 99% to 100%, glutamine (Q) at position 1 of SEQ No. 9 is modified to pyroglutamate, and / or, in more than 90% of the antibody, preferably about 95% to 100%, more preferably about 95% to 96%, lysine (K) at position 450 of SEQ No. 9 is absent. Claim 14 A composition according to any one of claims 1 to 13, wherein more than 85% of the glycan is fucosylated, preferably about 85% to 95% of the glycan is fucosylated. Claim 15 A composition according to any one of claims 1 to 14, wherein about 20% to 40% of the glycan is galactosylated. Claim 16 A composition according to any one of claims 1 to 15, wherein about 80% to 90% of the glycan is part of a major fucosylated glycan type. Claim 17 A composition according to any one of claims 1 to 16, wherein about 1% to 4% of the glycan is a mannose-containing glycan, preferably the glycan is of the Man5 and Man31F types. Claim 18 A composition according to any one of claims 1 to 17, wherein about 1% to 4%, preferably 1% to 3%, more preferably 1% to 2% of the glycan is of the Man5 type. Claim 19 A composition according to any one of claims 1 to 18, wherein less than 2% of the glycan is sialylated, and preferably about 0.5% to 2% of the glycan is sialylated. Claim 20 A composition according to any one of claims 1 to 19, wherein in one or more of the antibodies, the asparagine (N) at position 58, position 318, and / or position 387 of SEQ ID NO. 9 is deamidated. Claim 21 A composition according to any one of claims 1 to 20, wherein the asparagine (N) at position 58 of SEQ ID NO. 9 is deamidated in less than 5%, preferably less than 2%, preferably less than about 1%, preferably about 0% to 1% of the antibody. Claim 22 A composition according to any one of claims 1 to 21, wherein the asparagine (N) at position 318 of SEQ ID NO. 9 is deamidated in less than about 9%, preferably less than about 8%, preferably about 6% to 9% of the antibody. Claim 23 A composition according to any one of claims 1 to 22, wherein the asparagine (N) at position 387 of SEQ ID NO. 9 is deamidated in less than about 5%, preferably less than about 3%, preferably about 1% to 43% of the antibody. Claim 24 A composition according to any one of claims 1 to 23, wherein in one or more of the antibodies, the methionine at position 71, position 115, position 255, position 361, and / or position 431 of SEQ ID NO. 9 is oxidized. Claim 25 A composition according to any one of claims 1 to 24, wherein the methionine (M) at position 71 of SEQ ID NO. 9 is oxidized in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1% of the antibody. Claim 26 A composition according to any one of claims 1 to 25, wherein the methionine (M) at position 115 of SEQ ID NO. 9 is oxidized in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably about 1% to 3% of the antibody. Claim 27 A composition according to any one of claims 1 to 26, wherein the methionine (M) at position 255 of SEQ ID NO. 9 is oxidized in less than about 5%, preferably less than about 3%, preferably about 1% to 30% of the antibody. Claim 28 A composition according to any one of claims 1 to 27, wherein the methionine (M) at position 361 of SEQ ID NO. 9 is oxidized in less than about 5%, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1% of the antibody. Claim 29 A composition according to any one of claims 1 to 28, wherein the methionine (M) at position 431 of SEQ ID NO. 9 is oxidized in less than about 5%, preferably less than about 3%, preferably less than about 2%, preferably about 1% to 2% of the antibody. Claim 30 A composition according to any one of claims 1 to 29, wherein in one or more of the antibodies, the aspartate (D) at position 283 of sequence number 9 is modified into iso-aspartate. Claim 31 A composition according to any one of claims 1 to 30, wherein in one or more of the antibodies, the proline (P) at position 448 of SEQ ID NO. 9 is amidated. Claim 32 A composition according to any one of claims 1 to 31, wherein in one or more of the antibodies, glycine (G) at position 449 of sequence number 9 is absent. Claim 33 A composition according to any one of claims 1 to 32, wherein one or more of the antibodies are fragmented through clipping between the asparagine at position 58 and the threonine at position 59 of SEQ ID NO.

9. Claim 34 A composition according to any one of claims 1 to 33, wherein in one or more of the antibodies, the light chain (LC) and / or heavy chain (HC) is glycated, preferably (i) less than about 35% of the antibody, preferably less than about 2%, preferably about 1% to 3%, in which the LC is glycated and / or; or (ii) less than about 6% of the antibody, preferably less than about 5%, preferably about 2% to 5%, in which the HC is glycated. Claim 35 A composition according to any one of claims 1 to 32, wherein the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2). Claim 36 In claim 35, the antibody comprises one or more of the following disulfide crosslinks: composition: LC:C23-LC:C88; LC:C134-LC:C194; LC:C214-HC:C223; HC:C22-HC:C97; HC:C147-HC:C203; HC1:229-HC2:229 and HC1:232-HC2:232; HC:C264-HC:C324; and HC:C370-HC:C428 (wherein the numbering of the cysteine ​​residues (C) corresponds to their positions in sequence numbers 8 and 9). Claim 37 In any one of claims 1 to 36, the antibody has (i) a molecular weight of 147.0 to 147.6 kDa and / or; (ii) a pI of 8.4 (theoretical value) and 9.3 (determined value) for each, and / or; (iii) a quenching coefficient (A 0.1% A composition in which )(mL / (mg * cm)) is 1.39 (theoretical value) and 1.438 (experimentally determined value) for each. Claim 38 A composition according to any one of claims 1 to 37, wherein one or more antibodies are acidic variants. Claim 39 A composition according to any one of claims 1 to 38, wherein the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably CHO-K1 cells. Claim 40 A composition according to any one of claims 1 to 39, wherein the HC of the antibody further comprises a signal peptide derived from a human immunoglobulin heavy chain, and the LC further comprises a signal peptide derived from a human immunoglobulin kappa light chain, preferably the signal peptide of the HC has the amino acid sequence presented in SEQ ID NO. 17, and the signal peptide of the LC has the amino acid sequence presented in SEQ ID NO.

18. Claim 41 A composition according to claim 40, wherein the HC containing the signal peptide has the amino acid sequence presented in SEQ ID NO. 19, and the LC containing the signal peptide has the amino acid sequence presented in SEQ ID NO.

20. Claim 42 A composition comprising one or more antibodies, wherein the antibody comprises an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK (wherein, in one or more of the above antibodies, glutamine (Q) at position 1 is modified to pyroglutamate (pE), lysine (K) at position 450 is absent, and asparagine (N) at position 300 is glycosylated), where Sequence No. 8 is the following sequence Including:DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, where the antibody is of the following glycan type: Man3+1F,A composition comprising G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, wherein the antibodies are recombinant antibodies produced in Chinese hamster ovary (CHO)-K1 cells. Claim 43 An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, wherein SEQ No. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK,where Sequence No. 8 is the following sequence Includes:DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, where HC includes the following modifications: glutamine (Q) at position 1 is modified to pyroglutamate; lysine (K) at position 450 is deleted;Asparagine (N) at position 300 is glycosylated, wherein the antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2.; Claim 44 An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, wherein SEQ No. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK,where Sequence No. 8 is the following sequence Including:DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, where HC includes the following modifications: glutamine (Q) at position 1 is modified to pyroglutamate; lysine (K) at position 450 is deleted; and asparagine (N) at position 300 is glycosylated, wherein less than 3% of the glycans are of the Man5 type, an antibody. Claim 45 In paragraph 43 or 44, the antibody, wherein HC further comprises one or more of the following modifications: asparagine (N) at position 58 is deamidated; methionine (M) at position 71 is oxidized; methionine (M) at position 115 is oxidized; methionine (M) at position 255 is oxidized; aspartate (D) at position 283 is isomerized; asparagine (N) at position 318 is deamidated or contains succinimide; methionine (M) at position 361 is oxidized; asparagine (N) at position 387 is deamidated or contains succinimide; methionine (M) at position 431 is oxidized; glycine (G) at position 449 is absent; proline (P) at position 448 is amidated after loss of C-terminal lysine and glycine. Claim 46 An antibody according to any one of claims 43 to 45, wherein the major glycan types are G0F and G1F, preferably about 49% of the glycan is of the G0F type and about 25% of the glycan is of the G1F type. Claim 47 An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 7 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 7 comprises the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX5X6X7 (where X2 is absent or glutamine or pyroglutamate (pE); X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or glycine; X7 is absent or lysine;Herein, SEQ ID NO. 8 is an antibody comprising the following sequence: DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.; Claim 48 In claim 47, HC is an antibody having the amino acid sequence of SEQ ID NO. 39 comprising the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGX I TYDNPSLKSRLTX II SVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTX III VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVX IV GVEVHNAKTKPREEQYX V STYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEX VI TKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX VII HEALHNHYTQKSLSLSX5X6X7(wherein, X1 is absent or glutamine or pyroglutamate (pE); X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine, deamidated asparagine, or asparagine containing succinimide; X5 is proline or amidated proline; X6 is absent or glycine; X7 is absent or lysine; X I is asparagine or deamidated asparagine; X II is methionine or oxidized methionine and;X III is methionine or oxidized methionine; X IV is aspartate or iso-aspartate;X V is asparagine or glycosylated asparagine;X VI is methionine or oxidized methionine; X VII (is methionine or oxidized methionine). Claim 49 In paragraph 47 or 48, X1 is an antibody that is pyroglutamate (pE). Claim 50 In paragraph 47 or 48, X1 is an antibody that is glutamine. Claim 51 In any one of paragraphs 47 to 50, X7 is an antibody that is absent. Claim 52 In any one of paragraphs 47 to 50, X7 is an antibody that is lysine. Claim 53 In paragraph 51, X6 is an absent antibody. Claim 54 In any one of paragraphs 47 to 52, X6 is an antibody that is glycine. Claim 55 In any one of paragraphs 47 to 54, X5 is an antibody that is amidated proline. Claim 56 In any one of paragraphs 47 to 54, X5 is an antibody that is proline. Claim 57 In any one of paragraphs 47 to 56, X2 is an antibody that is oxidized methionine. Claim 58 In any one of paragraphs 47 to 56, X2 is methionine, an antibody. Claim 59 In any one of paragraphs 47 to 58, X3 is an antibody that is deamidated asparagine. Claim 60 In any one of paragraphs 47 to 58, X3 is an antibody comprising asparagine containing succinimide. Claim 61 In any one of paragraphs 47 to 58, X3 is an antibody that is asparagine. Claim 62 In any one of paragraphs 47 to 61, X4 is an antibody that is deamidated asparagine. Claim 63 In any one of paragraphs 47 to 61, X4 is an antibody comprising asparagine containing succinimide. Claim 64 In any one of paragraphs 47 to 61, X4 is an antibody that is asparagine. Claim 65 In any one of paragraphs 48 through 64, X I An antibody that is deamidated asparagine. Claim 66 In any one of paragraphs 48 through 64, X I It is an antibody that is asparagine. Claim 67 In any one of paragraphs 48 through 66, X II is an antibody that is oxidized methionine. Claim 68 In any one of paragraphs 48 through 66, X II is an antibody that is methionine. Claim 69 In any one of paragraphs 48 through 68, X III Antibody, which is oxidized methionine. Claim 70 In any one of paragraphs 48 through 68, X III An antibody that is methionine. Claim 71 In any one of paragraphs 48 through 70, X IV is an antibody that is iso-aspartate. Claim 72 In any one of paragraphs 48 through 70, X IV is an antibody that is aspartate. Claim 73 In any one of paragraphs 48 through 72, X V is an antibody that is glycosylated asparagine. Claim 74 In any one of paragraphs 48 through 72, X V is an antibody that is asparagine. Claim 75 In any one of paragraphs 48 through 74, X VI Antibody, which is oxidized methionine. Claim 76 In any one of paragraphs 48 through 74, X VI An antibody that is methionine. Claim 77 In any one of paragraphs 48 through 76, X VII Antibody, which is oxidized methionine. Claim 78 In any one of paragraphs 48 through 76, X VII An antibody that is methionine. Claim 79 In any one of paragraphs 47 to 78, X1 is pyroglutamate (pE), X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, and X7 is an absent antibody. Claim 80 In any one of paragraphs 48 to 79, X1 is pyroglutamate (pE), X2 is methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X I is asparagine, and X II is methionine, and X III is methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII is methionine; or X1 is pyroglutamate (pE), X2 is oxidized methionine, X3 is asparagine, X4 is asparagine, X5 is proline, X6 is glycine, X7 is absent, and X I is asparagine, and X II is methionine, and X III is oxidized methionine, and X IV is aspartate, and X V is glycosylated asparagine, and X VI is methionine, and X VII An antibody that is methionine. Claim 81 In any one of paragraphs 42 to 80, the antibody comprises a glycosylated light chain (LC). Claim 82 In any one of paragraphs 42 to 81, the antibody comprises a glycosylated heavy chain (HC). Claim 83 In any one of paragraphs 42 to 82, the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2). Claim 84 In claim 83, the antibody comprises one or more of the following disulfide crosslinks: antibody:LC:C23-LC:C88;LC:C134-LC:C194;LC:C214-HC:C223;HC:C22-HC:C97;HC:C147-HC:C203;HC1:229-HC2:229 and HC1:232-HC2:232;HC:C264-HC:C324; and HC:C370-HC:C428 (wherein the numbering of the cysteine ​​residue (C) corresponds to the positions thereof in sequence nos. 7 and 8 (wherein glutamine (X1) is present in sequence no. 7) and in sequence nos. 9 and 8, respectively). Claim 85 In any one of paragraphs 42 to 84, the antibody is an antibody having a molecular weight of 147.0 to 147.6 kDa. Claim 86 In any one of paragraphs 42 to 85, the antibody is an antibody having a pI of 8.4 (theoretical value) and 9.3 (experimentally determined value) for each. Claim 87 In any one of paragraphs 42 to 86, the antibody is a quenching coefficient (A 0.1% Antibodies with )(mL / (mg * cm)) values ​​of 1.39 (theoretical value) and 1.438 (experimentally determined value) for each. Claim 88 In any one of paragraphs 42 through 87, the antibody is an antibody that is an acidic antibody variant. Claim 89 An antibody according to any one of claims 42 to 88, wherein the constant region of each HC contains one N-linked glycan site at residue N300 as presented in SEQ ID NO. 7 or 9. Claim 90 In paragraph 89, the antibody comprises one or more of the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, preferably the major glycan types being G0F and G1F. Claim 91 An antibody according to claim 89 or 90, wherein more than 85% of the glycan is fucosylated, preferably about 85% to 95% of the glycan is fucosylated. Claim 92 An antibody according to any one of claims 89 to 91, wherein about 20% to 40% of the glycan is galactosylated. Claim 93 An antibody according to any one of claims 89 to 92, wherein about 80% to 90% of the glycan is part of a major fucosylated glycan type, preferably the major glycan types are G0F and G1F. Claim 94 An antibody according to any one of claims 89 to 94, wherein about 1% to 4% of the glycan is a mannose-containing glycan, preferably the glycan is of the Man5 and Man31F types. Claim 95 An antibody according to any one of paragraphs 89 to 94, wherein about 1% to 4% of the glycan is of the Man5 type. Claim 96 An antibody according to any one of claims 89 to 95, wherein less than about 3% of the glycan is of the Man5 type, preferably about 1% to 2.5% of the glycan, preferably about 1% to 2% is of the Man5 type. Claim 97 An antibody according to any one of claims 89 to 96, wherein less than 2% of the glycan is sialylated, preferably about 0.5% to 2% of the glycan is sialylated. Claim 98 In any one of paragraphs 42 to 87, the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably the antibody is an antibody produced in CHO-K1 cells. Claim 99 An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, wherein SEQ No. 9 comprises the following sequence: QLQLQESGPG LVKPSETLSL TCSVSGGSII SRSSYWGWIR QPPGKGLEWIGGIYHSGNTY DNPSLKSRLT MSVDTSKNQF SLNLRSVTAA DTAVYYCARIVPGGDAFDIW GQGTMVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKRVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK(wherein glutamine (Q) at position 1 is modified to pyroglutamate; lysine (K) at position 450 is deleted; and asparagine (N) at position 300 is glycosylated);wherein the antibody [is] the following disulfide crosslink Including:LC:C23-LC:C88;LC:C134-LC:C194;LC:C214-HC:C223;HC:C22-HC:C97;HC:C147-HC:C203;HC1:229-HC2:229 and HC1:232-HC2:232;HC:C264-HC:C324; and HC:C370-HC:C428 (wherein the numbering of cysteine ​​residues (C) corresponds to their positions in sequence numbers 8 and 9);The above antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, preferably the major glycan types are G0F and G1F; wherein SEQ No. 8 is an antibody comprising the following sequence: DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.; Claim 100 In paragraph 99, an antibody in which less than 3% of the glycan is of the Man5 type. Claim 101 In claim 99 or 100, the antibody, wherein HC further comprises one or more of the following modifications: asparagine (N) at position 58 is deamidated; methionine (M) at position 71 is oxidized; methionine (M) at position 115 is oxidized; methionine (M) at position 255 is oxidized; aspartate (D) at position 283 is isomerized; asparagine (N) at position 318 is deamidated or contains succinimide; methionine (M) at position 361 is oxidized; asparagine (N) at position 387 is deamidated or contains succinimide; methionine (M) at position 431 is oxidized; glycine (G) at position 449 is absent; proline (P) at position 448 is after the loss of C-terminal lysine and glycine Amidated; lysine (K) residues are glycosylated. Claim 102 An antibody according to any one of claims 42 to 101, wherein HC further comprises a signal peptide derived from a human immunoglobulin heavy chain, and LC further comprises a signal peptide derived from a human immunoglobulin kappa light chain, preferably the signal peptide of HC has the amino acid sequence presented in SEQ ID NO. 17, and the signal peptide of LC has the amino acid sequence presented in SEQ ID NO. 18, and preferably HC containing the signal peptide has the amino acid sequence presented in SEQ ID NO. 19, and LC containing the signal peptide has the amino acid sequence presented in SEQ ID NO.

20. Claim 103 An antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ ID NO. 7 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ ID NO. 8, wherein SEQ ID NO. 7 comprises the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGNTYDNPSLKSRLTMSVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSX5X6X7 (where X2 is absent or glutamine or pyroglutamate (pE); X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine or deamidated asparagine; X5 is proline or amidated proline; X6 is absent or glycine; X7 is absent or lysine;Herein, SEQ ID NO. 8 comprises the following sequence: DIQMTQSPSSLSASVGDRVTIACRASQSVGTYLNWYQQKRGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC, wherein HC further comprises a signal peptide derived from a human immunoglobulin heavy chain, and LC further comprises a signal peptide derived from a human immunoglobulin kappa light chain, preferably the signal peptide of HC has the amino acid sequence presented in SEQ ID NO. 17, and the signal peptide of LC has the amino acid sequence presented in SEQ ID NO. 18, an antibody.; Claim 104 In claim 103, HC is an antibody having the amino acid sequence of SEQ ID NO. 39 comprising the following sequence: X1LQLQESGPGLVKPSETLSLTCSVSGGSIISRSSYWGWIRQPPGKGLEWIGGIYHSGX I TYDNPSLKSRLTX II SVDTSKNQFSLNLRSVTAADTAVYYCARIVPGGDAFDIWGQGTX III VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX2ISRTPEVTCVVVDVSHEDPEVKFNWYVX IV GVEVHNAKTKPREEQYX V STYRVVSVLTVLHQDWLX3GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEX VI TKNQVSLTCLVKGFYPSDIAVEWESX4GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX VII HEALHNHYTQKSLSLSX5X6X7(wherein, X1 is absent or glutamine or pyroglutamate (pE); X2 is methionine or oxidized methionine; X3 is asparagine, deamidated asparagine, or asparagine containing succinimide; X4 is asparagine, deamidated asparagine, or asparagine containing succinimide; X5 is proline or amidated proline; X6 is absent or glycine; X7 is absent or lysine; X I is asparagine or deamidated asparagine; X II is methionine or oxidized methionine and;X III is methionine or oxidized methionine; X IV is aspartate or iso-aspartate;X V is asparagine or glycosylated asparagine;X VI is methionine or oxidized methionine; X VII (is methionine or oxidized methionine). Claim 105 An antibody according to claim 103 or 104, wherein the HC containing the signal peptide has the amino acid sequence presented in SEQ ID NO. 19, and the LC containing the signal peptide has the amino acid sequence presented in SEQ ID NO.

20. Claim 106 One or more polynucleotide(s) comprising a nucleotide sequence encoding an immunoglobulin heavy chain (HC) and an immunoglobulin light chain (LC) of an anti-transthyretin (TTR) antibody, wherein the HC has the amino acid sequence of SEQ ID NO. 7, 9, or 39, and the LC has the amino acid sequence of SEQ ID NO.

8. Claim 107 Nucleic acid molecules comprising: (a) a first nucleotide sequence presented in SEQ ID NO. 13 encoding the immunoglobulin heavy chain (HC) of an anti-transthyretin (TTR) antibody, and a second nucleotide sequence presented in SEQ ID NO. 14 encoding the immunoglobulin light chain (LC) of an anti-transthyretin (TTR) antibody; (b) a messenger RNA (mRNA) equivalent of the first and second nucleotide sequences of (a); (c) a polynucleotide(s) comprising a sequence complementary to the first and second nucleotide sequences of (a) or the mRNA equivalent thereof of (b); or (d) a polynucleotide(s) comprising a sequence that is a degenerate of the first and second nucleotide sequences of (a) or the mRNA equivalent thereof of (b). Claim 108 In claim 107, the sequence of the degenerate of the first nucleotide sequence is presented in SEQ ID NO. 15, and the sequence of the degenerate of the second nucleotide sequence is presented in SEQ ID NO. 16, a nucleic acid molecule. Claim 109 Polynucleotide(s) of claim 106 or nucleic acid molecule of claim 107 or 108, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from a human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from a human immunoglobulin kappa light chain, preferably the nucleotide sequence of the first signal peptide is presented in SEQ ID NO. 21 and the nucleotide sequence of the second signal peptide is presented in SEQ ID NO. 22, polynucleotide(s) or nucleic acid molecule. Claim 110 A polynucleotide(s) or nucleic acid molecule according to claim 109, wherein the first nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence presented in SEQ ID NO. 23, and the second nucleotide sequence comprising the nucleotide sequence of the signal peptide has the sequence presented in SEQ ID NO.

24. Claim 111 One or more polynucleotide(s) comprising a first nucleotide sequence encoding an immunoglobulin heavy chain (HC) of an anti-transthyretin (TTR) antibody and a second nucleotide sequence encoding an immunoglobulin light chain (LC), wherein the HC has the amino acid sequence of SEQ ID NO. 7, 9, or 39 and the LC has the amino acid sequence of SEQ ID NO. 8, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from a human immunoglobulin heavy chain and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from a human immunoglobulin kappa light chain, preferably wherein the nucleotide sequence of the first signal peptide is presented in SEQ ID NO. 21 and the nucleotide sequence of the second signal peptide is presented in SEQ ID NO.

22. Claim 112 A nucleic acid molecule comprising a polynucleotide(s) comprising a first nucleotide sequence presented in SEQ ID NO. 13 encoding an immunoglobulin heavy chain (HC) of an anti-transthyretin (TTR) antibody, and a second nucleotide sequence presented in SEQ ID NO. 14 encoding an immunoglobulin light chain (LC) of an anti-transthyretin (TTR) antibody, wherein the first nucleotide sequence comprises a nucleotide sequence encoding a first signal peptide derived from a human immunoglobulin heavy chain, and the second nucleotide sequence comprises a nucleotide sequence encoding a second signal peptide derived from a human immunoglobulin kappa light chain, preferably wherein the nucleotide sequence of the first signal peptide is presented in SEQ ID NO. 21 and the nucleotide sequence of the second signal peptide is presented in SEQ ID NO.

22. Claim 113 One or more expression vector(s) comprising a polynucleotide(s) of any one of claims 106 and 109 to 111, or a nucleic acid molecule of any one of claims 107 to 110 and 112. Claim 114 A host cell comprising the polynucleotide(s) of any one of claims 106 and 109 to 111, or the nucleic acid molecule of any one of claims 107 to 110 and 112, or the vector(s) of claim 114. Claim 115 In paragraph 114, non-human cells, preferably E. coli ( E. coli ), insect or CHO cell, host cell. Claim 116 In paragraph 114 or 115, a host cell, preferably a CHO cell, which is a CHO-K1 cell. Claim 117 A method for producing an anti-TTR antibody, comprising: (a) culturing a cell according to any one of claims 114 to 116; and (b) isolating the antibody or its immunoglobulin chain(s) from the culture medium. Claim 118 A method for generating an antibody of any one of claims 42 to 105 or an antigen-binding fragment thereof, wherein the antibody comprises at least the following post-translational modifications: glutamine (Q) at position 1 of X1 and SEQ No. 9 is each pyroglutamate (pE) and / or lysine (K) at position 450 of X7 and SEQ No. 9 is each absent; the method comprises: a) cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a first nucleotide sequence, into an expression vector; and cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a second nucleotide sequence, into an expression vector—wherein the first and second nucleotide sequences may be provided in the same or different expression vectors—; b) transforming the expression vector(s) into CHO-K1 cells; and c) an immunoglobulin chain comprising a heavy chain and a light chain. A method comprising: a step of culturing host cells under conditions that enable expression; d) isolating an immunoglobulin chain and a generated IgG antibody from the culture medium, respectively, and optionally, a step of generating a Fab fragment by using enzymatic digestion, such as papain cleavage, to produce an antigen-binding fragment of the antibody by digesting the IgG antibody. Claim 119 A method for generating an antibody of any one of claims 42 to 105 or an antigen-binding fragment thereof, wherein the antibody comprises at least the following post-translational modifications: glutamine (Q) at position 1 of sequence no. 9 is pyroglutamate (pE); lysine (K) at position 450 of sequence no. 9 is absent; asparagine (N) at position 300 of sequence no. 9 is glycosylated, wherein less than 3% of the glycans are of the Man5 type; the method comprises: a) cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a first nucleotide sequence, into an expression vector; and cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a second nucleotide sequence, into an expression vector—wherein the first and second nucleotide sequences may be provided in the same or different expression vectors—; b) expression A method comprising: a step of transforming vector(s) into CHO-K1 cells; c) culturing host cells under conditions that enable the expression of an immunoglobulin chain comprising a heavy chain and a light chain, wherein the culturing is performed with a pH dead band of 0.1 to 0.05, preferably 0.05; d) isolating the immunoglobulin chain and the generated IgG antibody from the culture medium, respectively, and optionally, generating a Fab fragment by using enzymatic degradation including papain cleavage to generate an antigen-binding fragment of the antibody by degrading the IgG antibody. Claim 120 An antibody that can be encoded by the polynucleotide(s) of any one of claims 106 and 109 to 111, can be encoded by the vector(s) of claim 113, or obtainable by the method of any one of claims 117 to 119. Claim 121 A composition comprising one or more of the antibodies of any one of claims 42 to 105 and 120. Claim 122 A composition according to claim 121, wherein in one or more of the antibodies, for each, glutamine (Q) at position 1 of SEQ No. 9 is modified to pyroglutamate, and X1 in SEQ No. 7 or 39 is pyroglutamate. Claim 123 A composition according to claim 121 or 122, wherein, in greater than 90% of the antibody, preferably about 99% to 100%, the glutamine (Q) at position 1 of SEQ No. 9 is modified to pyroglutamate for each, and X1 in SEQ No. 7 or 39 is pyroglutamate. Claim 124 A composition according to claim 121 or 122, wherein in one or more of the antibodies, lysine (K) at position 450 of sequence number 9 and X7 at sequence number 7 or 39 are absent for each. Claim 125 A composition according to any one of claims 121 to 124, wherein in more than 90% of the antibody, preferably about 95% to 100%, preferably about 95% to 96%, lysine (K) at position 450 of SEQ ID NO. 9 and X7 at SEQ ID NO. 7 or 39 are each absent. Claim 126 In any one of claims 121 to 125, the antibody comprises a glycan, preferably an N-glycan, and preferably, for each, the asparagine (N) at position 300 of SEQ ID NO. 9 is glycosylated, and X in SEQ ID NO. 39 V A composition that is glycosylated asparagine. Claim 127 A composition according to any one of claims 121 to 126, wherein the antibody comprises the following glycan types: Man3+1F, G0-GN, G0F-GN, G0, G0F, Man5, G1F-GN / G1a, G1b, G1Fa, G1Fb, G2F, G2FS1, and G2FS2, preferably the major glycan types being G0F and G1F. Claim 128 A composition according to claim 126 or 127, wherein more than 85% of the glycan is fucosylated, preferably about 85% to 95% of the glycan is fucosylated. Claim 129 A composition according to any one of claims 126 to 128, wherein about 20% to 40% of the glycan is galactosylated. Claim 130 A composition according to any one of claims 126 to 129, wherein about 80% to 90% of the glycan is part of a major fucosylated glycan type, preferably the major glycan types are G0F and G1F. Claim 131 A composition according to any one of claims 125 to 130, wherein about 1% to 4% of the glycan is a mannose-containing glycan, preferably the glycan is of the Man5 and Man31F types. Claim 132 A composition according to any one of claims 125 to 131, wherein less than about 3% of the glycan, preferably about 1% to 2.5%, preferably 1% to 2%, is of the Man5 type. Claim 133 A composition according to any one of claims 125 to 132, wherein less than 2% of the glycan is sialylated, preferably about 0.5% to 2% of the glycan is sialylated. Claim 134 In any one of claims 121 to 133, in one or more of the antibodies, for each, the asparagine (N) at position 58, position 318, and / or position 387 of SEQ ID NO. 9 is deamidated, and X in SEQ ID NO. 39 I A composition in which X3 and / or X4 are deamidated asparagine. Claim 135 In any one of claims 121 to 134, in less than 5% of the antibody, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1%, for each, the asparagine (N) at position 58 of SEQ ID NO. 9 is deamidated, and X in SEQ ID NO. 39 I A composition that is deamidated asparagine. Claim 136 A composition according to any one of claims 121 to 135, wherein less than about 9% of the antibody, preferably less than about 8%, preferably about 6% to 9%, for each, the asparagine (N) at position 318 of SEQ ID NO. 9 is deamidated, and X3 in SEQ ID NO. 39 is the deamidated asparagine. Claim 137 A composition according to any one of claims 121 to 136, wherein, in less than about 5% of the antibody, preferably less than about 3%, preferably about 1% to 4%, the asparagine (N) at position 387 of SEQ ID NO. 9 is deamidated for each, and X4 in SEQ ID NO. 39 is the deamidated asparagine. Claim 138 In any one of claims 121 to 137, in one or more of the antibodies, for each, the methionine at position 71, position 115, position 255, position 361, and / or position 431 of SEQ ID NO. 9 is oxidized, and X in SEQ ID NO. 39 II , X III , X2, X VI and / or X VII A composition that is oxidized methionine. Claim 139 In any one of claims 121 to 138, in less than about 5% of the antibody, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1%, for each, the methionine (M) at position M71 of SEQ ID NO. 9 is oxidized, and X in SEQ ID NO. 39 II A composition that is oxidized methionine. Claim 140 In any one of claims 121 to 139, at less than about 5% of the antibody, preferably less than about 3%, preferably less than about 2%, preferably about 0% to 2%, for each, the methionine (M) at position M115 of SEQ ID NO. 9 is oxidized, and X in SEQ ID NO. 39 III A composition that is oxidized methionine. Claim 141 A composition according to any one of claims 121 to 140, wherein less than about 5%, preferably less than about 3%, preferably about 1% to 4% of the antibody, for each, the methionine (M) at position M255 of SEQ ID NO. 9 is oxidized, and X2 in SEQ ID NO. 39 is oxidized methionine. Claim 142 In any one of claims 121 to 141, in less than about 5% of the antibody, preferably less than about 2%, preferably less than about 1%, preferably about 0% to 1%, for each, the methionine (M) at position M361 of SEQ ID NO. 9 is oxidized, and X in SEQ ID NO. 39 VI A composition that is oxidized methionine. Claim 143 In any one of claims 121 to 142, at less than about 5% of the antibody, preferably less than about 3%, preferably less than about 2%, preferably about 0% to 2%, for each, the methionine (M) at position M431 of SEQ ID NO. 9 is oxidized, and X in SEQ ID NO. 39 VII A composition that is oxidized methionine. Claim 144 In any one of claims 121 to 143, in one or more of the antibodies, for each, the aspartate (D) at position 283 of SEQ ID NO. 9 is modified to iso-aspartate, and X in SEQ ID NO. 39 IV A composition that is iso-aspartate. Claim 145 A composition according to any one of claims 121 to 144, wherein in one or more of the antibodies, for each, the proline (P) at position 448 of SEQ ID NO. 9 is amidated, and X5 of SEQ ID NO. 7 or 39 is amidated proline. Claim 146 A composition according to claim 121 or 145, wherein in one or more of the antibodies, glycine (G) at position 449 of sequence number 9 and X5 of sequence number 7 or 39 are absent for each. Claim 147 A composition according to any one of claims 121 to 146, wherein one or more of the antibodies are fragmented through clipping between the asparagine at position 58 and the threonine at position 59 of each of SEQ ID NO. 9 and SEQ ID NO. 7 or 39. Claim 148 A composition according to any one of claims 121 to 147, wherein in one or more of the antibodies, the light chain (LC) is saccharified. Claim 149 A composition according to any one of claims 121 to 148, wherein LC is saccharified at less than about 5%, preferably less than about 2%, preferably about 0% to 2% of the antibody. Claim 150 A composition according to any one of claims 121 to 149, wherein in one or more of the antibodies, the heavy chain (HC) is saccharified. Claim 151 A composition according to any one of claims 121 to 150, wherein less than about 6% of the antibody, preferably less than about 5%, preferably about 2% to 5%, the HC is saccharified. Claim 152 A composition according to any one of claims 121 to 151, wherein the antibody comprises a first HC (HC1), a first LC (LC1), a second HC (HC2), and a second LC (LC2). Claim 153 In claim 152, the antibody comprises one or more of the following disulfide crosslinks: composition: LC:C23-LC:C88; LC:C134-LC:C194; LC:C214-HC:C223; HC:C22-HC:C97; HC:C147-HC:C203; HC1:229-HC2:229 and HC1:232-HC2:232HC:C264-HC:C324; and HC:C370-HC:C428 (wherein the numbering of the cysteine ​​residues (C) corresponds to their positions in sequence numbers 8 and 9). Claim 154 In any one of claims 121 to 153, the antibody has (i) a molecular weight of 147.0 to 147.6 kDa and / or; (ii) a pI of 8.4 (theoretical value) and 9.3 (determined value) for each, and / or; (iii) a quenching coefficient (A 0.1% A composition in which )(mL / (mg * cm)) is 1.39 (theoretical value) and 1.438 (determined value) for each. Claim 155 A composition according to any one of claims 121 to 154, wherein the composition comprises acidic species of antibodies, preferably about 25% to 33% of acidic antibody species. Claim 156 In any one of claims 121 to 155, the composition comprises the following charge variants of the antibody: main peak ≥ 63.0%, acidic peak ≤ 32.0%, basic peak ≤ 5.0%. Claim 157 A composition according to any one of claims 121 to 156, wherein the antibody is a recombinant antibody produced in Chinese hamster ovary (CHO) cells, preferably CHO-K1 cells. Claim 158 The composition of claim 157, wherein the method comprises the step of culturing CHO-K1 cells to a pH dead band of less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ 0.075, preferably ≤ 0.05, more preferably 0.05 or 0.1, most preferably 0.

05. Claim 159 A composition according to any one of claims 121 to 158, wherein the HC of the antibody further comprises a signal peptide derived from a human immunoglobulin heavy chain, and the LC further comprises a signal peptide derived from a human immunoglobulin kappa light chain, preferably the signal peptide of the HC has the amino acid sequence presented in SEQ ID NO. 17, and the signal peptide of the LC has the amino acid sequence presented in SEQ ID NO.

18. Claim 160 A composition according to claim 159, wherein the HC containing the signal peptide has the amino acid sequence presented in SEQ ID NO. 19, and the LC containing the signal peptide has the amino acid sequence presented in SEQ ID NO.

20. Claim 161 A composition according to any one of claims 1 to 41 and 121 to 160, comprising: an antibody 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 0.5% (w / v); and water for injection, wherein the formulation has a pH of about 5.3 to about 6.

3. Claim 162 A pharmaceutical composition comprising an antibody at a concentration of about 50 mg / ml to about 100 mg / ml in an aqueous formulation for use in a method for treating TTR amyloidosis (ATTR) in a subject, wherein the formulation comprises, as excipients, L-histidine, L-histidine monohydrochloride, sucrose, polysorbate 80 (PS80), and water for injection. Claim 163 A composition of claim 161, or a composition for use according to claim 162, wherein the pH of the formulation is about 5.

8. Claim 164 A composition of claim 161 or 163, or a composition for use according to claim 162 or 163, wherein the formulation is a composition having an osmolal concentration ≥ 240 mOsm / Kg. Claim 165 A composition of claim 161, 163, or 164, or a composition for use according to any one of claims 162 to 164, wherein the formulation has a shelf-life of 24 months at 2 to 8°C under conditions protected from light. Claim 166 A composition according to any one of claims 161 and 163 to 165, or a composition for use according to any one of claims 162 to 165, wherein the formulation comprises an antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v, wherein the formulation has a pH of about 5.

8. Claim 167 A composition according to any one of claims 161 and 163 to 166, or a composition for use according to any one of claims 162 to 166, wherein the formulation is essentially composed of 50 mg or 100 mg of antibody, 2.12 mg of L-histidine, 5.56 mg of L-histidine monohydrochloride, 160 mg of sucrose, 0.6 mg of PS80, and water for injection, so that the total volume is about 2 ml. Claim 168 A composition according to any one of claims 161 and 163 to 167, or a composition for use according to any one of claims 162 to 167, wherein the antibody is produced in Chinese hamster ovary (CHO) cells, preferably in CHO cell line K1. Claim 169 A composition according to any one of claims 161 and 163 to 168, or a composition for use according to any one of claims 162 to 168, wherein the formulation is suitable for intravenous administration. Claim 170 A composition according to any one of claims 161 and 163 to 169, or a composition for use according to any one of claims 162 to 169, wherein the formulation is provided in a 2 mL or 20 mL single-dose vial. Claim 171 A composition according to any one of claims 161 and 163 to 170, or a composition for use according to any one of claims 162 to 170, wherein the formulation is provided in a 2 mL glass vial having an aluminum flip-off seal over a rubber stopper. Claim 172 A composition according to any one of claims 161, and 163 to 171, or a composition for use according to any one of claims 162 to 171, wherein the formulation is a preservative-free concentrate for an infusion solution provided as a sterile, colorless to slightly yellowish, transparent to slightly milky liquid that is essentially free of visible particles. Claim 173 A composition for said use, wherein, in any one of claims 162 to 172, the formulation is an intravenous infusion solution diluted in sterile glucose prior to administration. Claim 174 A pharmaceutical container comprising the composition of any one of claims 161, and 163 to 172. Claim 175 In paragraph 174, a pharmaceutical container which is a 2 ml or 20 ml vial. Claim 176 A vial of claim 175, a glass vial equipped with an aluminum flip-off cap over a 13 mm rubber stopper. Claim 177 A vial of claim 175 or 176 containing a pharmaceutical formulation with approximately 12.5% ​​volume overfill, or a total volume of 2.25 ml or 22.5 ml. Claim 178 A stable aqueous formulation for use in a method for treating TTR amyloid cardiomyopathy (ATTR-CM), comprising essentially (i) (a) an anti-TTR antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 7 or 39 or SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, (b) histidine at a concentration of about 20 mM, (c) sucrose at a concentration of about 8% (w / v), (d) PS80 at a concentration of about 0.03% (w / v), and (e) water for injection, (ii) having a pH of about 5.8; (iii) contained in a 2 mL single-use vial containing about 100 mg of antibody or a 20 mL single-use vial containing 1000 mg of antibody; (iv) diluted with a sterile glucose solution before administration to a concentration of at least 1 mg / mL of antibody; and (v) administered by intravenous infusion, Stable aqueous formulation. Claim 179 A formulation for the use of claim 178, wherein the N-terminal glutamine of the HC amino acid sequence is converted to pyroglutamate and the C-terminal lysine of the HC amino acid sequence is deleted, wherein HC is N-glycosylated. Claim 180 In claim 179, HC is a formulation for the above use having the amino acid sequence of SEQ ID NO.

15. Claim 181 A therapeutic kit comprising (i) one or more containers of any one of claims 174 to 177; (ii) means for delivering a formulation to a human subject, wherein the means particularly comprises an infusion bag or syringe for intravenous administration of an antibody. Claim 182 In claim 181, the formulation is a preservative-free, clear to milky white, colorless to pale yellow solution provided in a vial of 100 mg / 2 mL, a therapeutic kit. Claim 183 A manufactured article comprising: (i) one or more container(s) of any one of claims 174 through 177, and (ii) a label—wherein the label specifies that the antibody is indicated for the treatment of ATTR, particularly wild-type or hereditary transthyretin-mediated amyloid cardiomyopathy (ATTR-CM)—and / or (iii) an accompanying document specifying that the antibody must be administered intravenously. Claim 184 In paragraph 183, the manufactured article comprises a container including a 2 mL or 20 mL glass vial. Claim 185 Article 183 or 184, wherein the antibody in the container is diluted prior to administration, wherein the dilution is performed using a 5% glucose solution, and optionally the glucose solution for dilution is provided in a second container within the article. Claim 186 An article manufactured according to any one of claims 183 to 185, wherein the antibody formulation is a preservative-free, clear to milky white, colorless to pale yellow solution provided in a vial at 100 mg / 2 mL. Claim 187 A method for treating TTR amyloidosis (ATTR), preferably ATTR cardiomyopathy (ATTR-CM) and / or ATTR polyneuropathy (ATTR-PN), comprising the step of administering to a patient in need of the antibody of any one of claims 42 to 105 and 119, or a composition of any one of claims 1 to 41, 121 to 161, and 163 to 171. Claim 188 In claim 187, the method comprises the step of administering an antibody to a patient having ATTR-CM to treat ATTR-CM in said patient. Claim 189 In claim 187, the method comprises the step of administering an antibody to a patient having ATTR-PN to treat ATTR-PN in said patient. Claim 190 A method according to any one of claims 187 to 189, wherein the antibody is diluted and administered as an intravenous infusion. Claim 191 In paragraph 190, the method is performed by diluting with a 5% glucose solution. Claim 192 A method according to any one of paragraphs 187 to 191, wherein the patient has previously been treated with a TTR tetramer stabilizer and / or is currently being provided with it in parallel. Claim 193 In paragraph 192, the TTR tetramer stabilizer is selected from tapamidis and acoramidis. Claim 194 The use of any one of the antibodies of claims 42 to 105 and 119 or any one of the compositions of claims 1 to 41, 121 to 161, and 163 to 171 in the manufacture of a drug for treating TTR amyloidosis (ATTR). Claim 195 In any one of paragraphs 42 to 105 and 119, the antibody is produced in CHO cells, preferably CHO-K1 cells, and is designated as ALXN2220. Claim 196 A composition for said use, wherein, in any one of claims 162 to 173, the subject has been treated with or is being treated concurrently with a TTR tetramer stabilizer, preferably tafamidis or acoramidis. Claim 197 A method for generating an antibody of any one of claims 42 to 105 and 109 or an antigen-binding fragment thereof, comprising: a) cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a first nucleotide sequence, into an expression vector; and cloning a nucleic acid molecule of any one of claims 107 to 110 and 112, comprising a second nucleotide sequence, into an expression vector—wherein the first and second nucleotide sequences may be provided in the same or different expression vectors—; b) transforming the expression vector(s) into non-human host cells, preferably CHO cells, more preferably CHO-K1 cells; c) culturing the host cells under conditions that enable the expression of an immunoglobulin chain comprising a heavy chain and a light chain; d) isolating the immunoglobulin chain and the generated IgG antibody from the culture medium, respectively, and optionally, digesting the IgG antibody to generate an antigen-binding fragment of the antibody, e.g. A method comprising: a step of generating a Fab fragment using enzymatic digestion including papain cleavage; and optionally e) a step of forming the antibody into an aqueous solution comprising an antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v (wherein the formulation has a pH of about 5.8), thereby producing a pharmaceutical composition comprising the antibody; and f) optionally, a step of filling the composition into a vial; and g) further optionally, a step of packaging the pharmaceutical composition and the vial, respectively, in a kit, together with instructions for administration of the antibody in a human patient, e.g., intravenous administration. Claim 198 In claim 197, step (c) comprises the step of culturing cells to a pH dead band of less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ 0.075, preferably ≤ 0.05, more preferably 0.05 or 0.1, most preferably 0.

05. Claim 199 A method for producing an antibody composition according to any one of claims 1 to 42 and claims 121 to 173, comprising: a) cloning a nucleic acid molecule according to any one of claims 107 to 110 and 112, comprising a first nucleotide sequence, into an expression vector; and cloning a nucleic acid molecule according to any one of claims 107 to 110 and 112, comprising a second nucleotide sequence, into an expression vector—wherein the first and second nucleotide sequences may be provided in the same or different expression vectors—; b) transforming the expression vector(s) into CHO-K1 cells; and c) culturing CHO-K1 cells under conditions enabling the expression of an immunoglobulin chain comprising a heavy chain and a light chain, wherein the culture is less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ -;d) a step of preparing a composition by isolating the immunoglobulin chain and the generated IgG antibody from the culture medium, respectively, and optionally, generating a Fab fragment by using enzymatic digestion including papain cleavage to generate an antigen-binding fragment of the antibody by digesting the IgG antibody; and optionally e) formulating the antibody into an aqueous solution comprising an antibody at a concentration of about 50 mg / ml or about 100 mg / ml, histidine at a concentration of about 20 mM, sucrose at a concentration of about 6.5% by weight / volume (w / v) or about 8% (w / v) sucrose, and PS80 at a concentration of about 0.03% w / v (wherein the formulation has a pH of about 5.8), a step of producing a pharmaceutical composition comprising an antibody; and f) optionally, a step of filling the composition into a vial; and g) additionally optionally, a step of packaging the pharmaceutical composition and the vial, respectively, in a kit, together with instructions for administering the antibody to a human patient, e.g., intravenous administration. Claim 200 A method for preparing an antibody of any one of claims 42 to 105 and 120, or a composition of any one of claims 1 to 42 and 121 to 173, comprising: (a) a step of culturing a cell of any one of claims 114 to 116—wherein the culturing is performed with a pH dead band of less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ 0.075, preferably ≤ 0.05, more preferably 0.05 or 0.1, most preferably 0.05—; and (b) a step of isolating the antibody or its immunoglobulin chain(s) from the culture medium. Claim 201 A method for verifying an amyloid-depleting drug comprises: (i) incubating a tissue section containing amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of an amyloid-depleting drug—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye—; and (ii) performing high-resolution live cell imaging, wherein, (a) the superposition of the fluorescent signal of the amyloid-depleting drug and the fluorescent signal of the amyloid indicates binding of the amyloid-depleting drug to the amyloid; (b) the presence of an intracellular fluorescent signal to a punctuate in a phagocytic vesicle indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of the punctuate fluorescent signal from the fluorescent signal of the amyloid indicates macrophage-mediated amyloid fragmentation. Claim 202 In claim 201, items (a) to (c) are a method in which tissue sections having amyloid deposits and macrophages are not cultured in the presence of an amyloid-depleting drug, but are not observed in control samples cultured in the presence of a control compound that does not bind to amyloid and / or cannot mediate phagocytosis. Claim 203 In claim 201 or 202, the amyloid-depleting drug, or a pharmaceutical composition comprising an amyloid-depleting drug, is a method used for the treatment of amyloidosis or amyloid-related diseases. Claim 204 A screening method for identifying and selectively obtaining amyloid-depleting drugs from a plurality of test compounds, comprising: (i) incubating a tissue section having amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of a test compound—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the test compound is labeled with a fluorescent dye—; The method comprises the step of (ii) performing high-resolution live cell imaging, wherein (a) the superposition of the fluorescent signal of the test compound and the fluorescent signal of the amyloid indicates binding of the test compound to the amyloid; (b) the presence of the intracellular fluorescent signal as a dot in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) the sequential separation of the dot fluorescent signal from the fluorescent signal of the amyloid indicates macrophage-mediated amyloid fragmentation, wherein the presence of any one of items (a) to (c) indicates suitability of the test compound as an amyloid-depleting drug, and optionally, the step of using the amyloid-depleting drug, or a pharmaceutical composition comprising the amyloid-depleting drug, for the treatment of amyloidosis or an amyloid-related disease. Claim 205 A method for analyzing the effect of an agent on the amyloid depletion activity of an amyloid depletion drug, comprising: (i) incubating a sample of a tissue section having amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of an amyloid depletion drug and an agent; and iii) a step of performing high-resolution live cell imaging of each sample, wherein, (a) the superposition of the fluorescence signal of the amyloid-depleting drug and the amyloid fluorescence signal indicates the binding of the amyloid-depleting drug to amyloid; (b) the presence of a dot-like intracellular fluorescence signal in phagocytic vesicles indicates macrophage-mediated amyloid internalization; and (c) a sequential separation of the dot-like fluorescence signal from the amyloid fluorescence signal indicates macrophage-mediated amyloid fragmentation, wherein, when comparing a tissue section with amyloid deposits and macrophages incubated in the presence of the amyloid-depleting drug but without the agent with a control sample, the substantially unchanged fluorescence pattern of items (a) to (c) indicates the suitability of the combination of the amyloid-depleting drug and the agent in the treatment of amyloidosis or amyloid-related diseases, and the substantially changed fluorescence pattern of any one of items (a) to (c) indicates the effect of the agent on the amyloid-depleting activity of the amyloid-depleting drug. Optionally, (a) enhanced fluorescence signal; (b) enhanced intracellular fluorescence signal in phagocytic vesicles; and (c) enhanced separation of punctate fluorescence signal from the fluorescence signal of amyloid indicates a synergistic effect on the amyloid depletion activity of the amyloid depletion drug, and (a) reduced fluorescence signal; (b) reduced intracellular fluorescence signal in phagocytic vesicles; and (c) reduced separation of punctate fluorescence signal from the fluorescence signal of amyloid indicates a detrimental effect on the amyloid depletion activity of the amyloid depletion drug, method. Claim 206 A method for screening amyloid-depleting drugs for their ability to bind to amyloid, mediate macrophage recruitment to amyloid deposits, and then perform amyloid fragmentation and internalization, comprising: (i) incubating a tissue section containing amyloid deposits with macrophages, preferably THP-1 derived macrophages, in the presence of an amyloid-depleting drug—wherein the tissue section is stained with an amyloid-specific fluorescent dye and / or the amyloid-depleting drug is labeled with a fluorescent dye—; and (ii) a step of performing high-resolution live cell imaging, wherein, (a) the superposition of the fluorescent signal of the amyloid-depleting drug and the fluorescent signal of the amyloid indicates binding of the amyloid-depleting drug to the amyloid; (b) the presence of the intracellular fluorescent signal as a dot in phagocytic vesicles indicates macrophage-mediated amyloid internalization; (c) the sequential separation of the dot fluorescent signal from the fluorescent signal of the amyloid indicates macrophage-mediated amyloid fragmentation; optionally, a step of using the amyloid-depleting drug, or a pharmaceutical composition comprising the amyloid-depleting drug, for the treatment of amyloidosis or an amyloid-related disease. Claim 207 A method according to any one of claims 201 to 206, wherein high-resolution live cell imaging uses refractive index imaging for cell visualization and fluorescence microscopy for amyloid imaging. Claim 208 A method according to any one of claims 201 to 207, wherein the amyloid-depleting drug is labeled with a fluorescent dye different from the fluorescent dye used to stain amyloid deposits. Claim 209 A method according to any one of claims 201 to 208, wherein the tissue section is obtained from a patient suffering from amyloidosis or an amyloid-related disease. Claim 210 A method according to any one of claims 205 to 209, wherein the amyloidosis or amyloid-related disease is transthyretin (TTR) amyloidosis or TTR amyloid-related disease, preferably cardiac TTR amyloidosis. Claim 211 A method according to any one of claims 201 to 210, wherein the amyloid-depleting drug is an anti-amyloid antibody or comprises an anti-amyloid antibody fragment. Claim 212 A method according to any one of claims 201 to 211, wherein the amyloid-depleting drug is an anti-TTR antibody or comprises an anti-TTR antibody fragment and / or the control compound is a homologous control antibody. Claim 213 A method according to claim 210 or 212, wherein the antibody is a human-derived, preferably human memory B cell-derived antibody, or a variant thereof comprising a heterogeneous constant domain, or a humanized antibody, or a chimeric antibody, and preferably the antibody is an IgG1 antibody, e.g., IgG1, λ antibody or IgG1, κ antibody. Claim 214 A method according to any one of claims 205 to 213, wherein the agent is a second drug for treating amyloidosis or an analgesic, and preferably the second drug is a TTR tetramer stabilizer or a silencer. Claim 215 A method for producing a pharmaceutical composition of an amyloid-depleting drug, comprising: (i) optionally producing an amyloid-depleting drug; (ii) applying the amyloid-depleting drug to the method of any one of claims 201 or 202 and 205 to 214; (iii) using the information obtained in step (ii) as part of an evaluation of whether the amyloid-depleting drug can be used as a pharmaceutical composition; and optionally, (iv) formulating the amyloid-depleting drug found to be useful as a pharmaceutical composition in step (iii) using a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier preferably comprises a buffer, a tonicity agent and / or a surfactant, most preferably all three components; and optionally (v) using the pharmaceutical composition for the treatment of amyloidosis or an amyloid-related disease. Claim 216 A method for characterizing, verifying, developing, and / or quality controlling an amyloid-depleting drug, comprising: (i) optionally generating an amyloid-depleting drug; (ii) applying the amyloid-depleting drug to the method of any one of claims 201 or 202 and 207 through 214; (iii) transmitting the information obtained in (ii) to a customer, contracting party, or cooperating partner and / or selecting a drug determined to be a suitable amyloid-depleting drug; and optionally, (iv) using the amyloid-depleting drug, or a pharmaceutical composition comprising the amyloid-depleting drug, for the treatment of amyloidosis or an amyloid-related disease. Claim 217 A kit useful for carrying out the method of any one of claims 201 to 216, comprising a first fluorescent dye for amyloid staining and / or a second fluorescent dye for labeling an amyloid-depleting drug, preferably comprising a negative and / or positive control, optionally comprising instructions for use together with the kit, preferably the positive control being an antibody of any one of claims 42 to 105 and 120. Claim 218 In any one of claims 201 to 214, the method is performed in addition to a method using a patient-derived amyloid xenograft (PDAX) non-human animal model, wherein the animal is characterized by an implant of amyloid fibrils derived from the tissue or organ of a patient suffering from amyloidosis or an amyloid-related disease, wherein the amyloid and amyloid fibrils each contain amyloid transthyretin (ATTR), and the amyloid fibrils are implanted subcutaneously or subcapsularly, or implanted in a kidney, peritoneum, muscle, brain, ventricles, nerve, eye, tongue, or heart, and the method comprises: (a) administering an amyloid-depleting drug or test substance to a PDAX non-human animal model; and (b) a step of determining amyloid fibrils in a model, wherein, upon administration of a drug or test substance, the accelerated removal or reduction of amyloid fibrils relative to a control group indicates the suitability of an amyloid-depleting drug for the treatment of amyloidosis or amyloid-related diseases and the amyloid-depleting activity of a test substance, respectively. Claim 219 A method in which, in any one of claims 201 to 216 and 218, the amyloid-depleting drug is the antibody of any one of claims 42 to 105 and 120. Claim 220 A method for producing an antibody comprising an immunoglobulin heavy chain (HC) having the amino acid sequence of SEQ No. 9 and an immunoglobulin light chain (LC) having the amino acid sequence of SEQ No. 8, comprising: (a) culturing host Chinese hamster ovary K1 (CHO-K1) cells comprising one or more expression vectors or vector systems comprising polynucleotides encoding antibodies HC and LC under conditions sufficient to express antibodies in a culture medium; (b) setting a pH set point and a pH dead band of the culture medium to adjust the levels of acidic, basic, and / or neutral antibody species in the culture medium; and (c) isolating antibodies HC and LC from the cell culture medium; and (d) optionally, formulating the isolated antibodies into a pharmaceutical formulation. Claim 221 A method according to claim 220, wherein step (b) comprises setting the pH set point to about 6.90 and the pH dead band to less than 0.2, preferably ≤ 0.175, preferably ≤ 0.15, preferably ≤ 0.125, preferably ≤ 0.1, preferably ≤ 0.075, preferably ≤ 0.05, more preferably 0.05 to 0.1 to reduce the level of acidic antibody species, and preferably setting the pH dead band to about 0.

05. Claim 222 A method for reducing the level of acidic species in a cultured antibody preparation compared to an antibody preparation produced using the same production method, except that the implementation of step (b) is implemented in a control group, e.g., in a pH dead band of pH 6.90 and about 0.

20. Claim 223 A method according to claim 221 or 222, wherein the implementation of step (b) reduces the level of N-linked mannose-5 glycan (Man5); preferably, the reduction in the Man5 level is greater than >20%; more preferably, the reduction in the Man5 level is about 40% to 50% compared to an antibody preparation produced using the same production method, except that step (b) is implemented at pH 6.90 and about 0.

20. Claim 224 A method according to any one of claims 220 to 223, wherein step (b) is implemented in a generating step comprising 5 to 14 days of the culture process, and is implemented between 5 to 14 days after seeding of host cells, for example, CHO-K1 cells. Claim 225 In any one of claims 220 to 224, step (b) is a method for controlling high mannose species in an antibody formulation without causing incidental adverse effects on secondary quality attributes selected from: (1) a relative level (%) of major antibody species vs. high molecular weight (HMW) vs. low molecular weight (LMW) antibody species in the formulation, as determined, for example, by size exclusion chromatography (SEC); (2) a relative level (%) of major antibody species in the formulation, as determined, for example, using capillary electrophoresis sodium dodecyl sulfate (CE-SDS-NR) under non-reducing conditions; and / or (3) a relative level (%) of antibody heavy chains and light chains in the formulation, as determined, for example, using capillary electrophoresis sodium dodecyl sulfate (CE-SDS-R) under reducing conditions. Claim 226 An antibody produced using the production method of any one of claims 220 to 225. Claim 227 In paragraph 226, the antibody comprises the heavy chain and light chain of ALXN2220 / NI006.

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