Stable Composition of Fc Multimers

Abstract

The present invention particularly relates to Fc multimer compositions, Fc multimer compositions comprising the Fc multimer compositions of the present invention in lyophilized form, and Fc multimer compositions according to the present invention for use in the treatment of autoimmune or inflammatory diseases.

Description

[Technical Field]

[0001] The present invention relates, in particular, to Fc polymer compositions, Fc polymer compositions comprising the Fc polymer composition of the present invention in a lyophilized form, and to the Fc polymer composition described in the present invention for use in the treatment of autoimmune diseases or inflammatory diseases. [Background technology]

[0002] Immunoglobulins play a vital role in the mammalian immune system. They are produced by B lymphocytes and found in plasma, lymph, and other bodily secretions. The basic unit of an immunoglobulin is a heterotetramer containing two heavy chains and two light chains linked by disulfide bonds. Each of these chains has a variable region at its N-terminus that forms an antigen-binding site, and a constant region that carries out the effector function of the immunoglobulin.

[0003] There are five major classes of immunoglobulins with distinct biochemical and physiological properties: IgG (γ-heavy chain), IgA (α), IgM (μ), IgD (δ), and IgE (ε). Human IgG is the most abundant immunoglobulin in plasma, while IgA is the dominant antibody class in exocrine secretions such as saliva, tears, and mucus from the respiratory and intestinal tracts. IgM is the physically largest antibody in the human circulatory system, usually existing as a pentamer of the basic immunoglobulin unit, and appears early in infection.

[0004] The Fc region (which is fragment crystallizable) is the tail region of immunoglobulins, and in the case of IgG and IgA, it contains two C-terminal domains of the heavy chain, and in the case of IgM and IgE, it contains three C-terminal domains of the heavy chain.

[0005] IgG products purified from human plasma, typically administered intravenously (IVIG) or subcutaneously (SCIG), are currently used in many clinical applications. In addition to their traditional uses for the treatment of primary or acquired immunodeficiency disorders and infectious diseases, these products have also been shown to be effective in treating autoimmune diseases and certain neurological disorders such as CIDP.

[0006] The mechanism of action of IVIG / SCIG products in the treatment of autoimmune diseases, inflammatory disorders, and neurological disorders appears to be primarily based on the effector function of IgG molecules mediated by the Fc region. The Fc region is responsible for interactions with Fc receptors and the complement system, thereby possessing immunomodulatory activity.

[0007] Fc receptors are receptors on the outer surface of immune system cells, such as phagocytes like human platelets, mast cells, macrophages, and monocytes, granulocytes like neutrophils, basophils, and eosinophils, and lymphocytes of the innate immune system (natural killer cells) or the adaptive immune system (e.g., B cells). Binding of the Fc region of an antibody via its Fab region, which adheres to an antigen (such as a pathogenic microorganism), typically results in the attachment of the pathogen to immune system cells. This, in turn, elicits a coordinated action against the pathogen.

[0008] The complement system is part of the immune system that attacks the cell membrane of pathogens by forming transmembrane channels that disrupt the pathogen's cell membrane. This subsequently leads to cell lysis and the death of the pathogen. Furthermore, activation of the complement system leads to inflammation, which stimulates phagocytic cells that eliminate foreign and damaging substances, as well as attracts further phagocytic cells.

[0009] Several strategies are being investigated to replace IVIG / SCIG for the treatment of autoimmune, inflammatory, and neurological disorders. One approach is to use only the Fc moiety of high concentrations of IgG, or to assemble several Fc moieties into a single molecule by recombination, for example, to increase the binding activity of the Fc moieties to Fc receptors. For example, various arrangements of multimeric Fc molecules were disclosed in Patent Document 1. Different advantageous constructs with arrangements of 2 to 10 Fc moieties were disclosed (see Momenta patent application, Patent Document 2). The most preferred option was a trivalent human IgG1 Fc molecule produced from two long chains of two Fc polypeptides linked by a mobile peptide linker and two short chains of a single Fc polypeptide. Mutations such as knob-in-hole mutations and electrostatic mutations were introduced to guide the construct toward correct assembly. Another example is a hexamer Fc molecule assembled by adding the 18-amino acid tail of the C-terminus of IgM to the Fc polypeptide, as disclosed, for example, in Patent Documents 3 and 4.

[0010] For therapeutic use, stable formulations of such Fc polymers are required. However, even single Fc fragments are highly susceptible to physical and chemical degradation, and are particularly prone to aggregation in solution. This is even more problematic with respect to Fc polymers. The first attempts to formulate Fc polymers in liquid form were only possible at very low protein concentrations (30 mg / ml). For therapeutic use, more concentrated solutions of these proteins are strongly desired. Stable compositions with suitable high protein concentrations have not yet been achieved, mainly because, as mentioned above, Fc polymers tend to aggregate in solution, and increasing the protein concentration increases the rate of aggregation, resulting in visible precipitates. It is known that such precipitates can be harmful when administered to patients. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] WO2008 / 151088A2 [Patent Document 2] WO2015 / 168643A2 [Patent Document 3] WO2014 / 060712A1 [Patent Document 4] WO2017 / 129737A1 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] The object of the present invention is thus to provide a stable Fc multimer composition having a higher concentration of Fc multimer, for example, enabling convenient administration of the Fc multimer to a patient by subcutaneous administration. [Means for Solving the Problems]

[0013] An Fc multimer composition comprising: a) an Fc multimer at a concentration between 60 mg / ml and 180 mg / ml, b) a pH value between 4.8 and 6.0, and c) a stabilizer at a concentration between 200 mM and 450 mM is provided.

[0014] The present invention further relates to an Fc multimer composition comprising the Fc multimer composition of the present invention in lyophilized form.

[0015] Furthermore, an Fc multimer composition of the present invention for use in the treatment of autoimmune or inflammatory diseases is provided. [Brief Description of the Drawings]

[0016] [Figure 1] It is a diagram showing the soluble (physical) aggregation (%HMW) over time at 35°C of Formulations 1 to 12. [Figure 2] It is a plot of pooled data of the rate of aggregation at 25°C and 35°C as a function of the measured pH. [Figure 3]This figure shows the time-dependent fragmentation (%LMW species) (NE-Caliper) of formulations 1-12 at 35°C. [Figure 4] This figure plots pooled data of the fragmentation rate (±SE) at 35°C as a function of measured pH. [Figure 5] This figure shows the time course of acid species formation (CEX) at 35°C for all formulations. [Figure 6] This figure plots pooled data of the rate of acidic species formation at 25°C and 35°C as a function of measured pH. [Figure 7] This figure shows the analysis of particles invisible to the naked eye. (a) Particle count (particles / mL) of particles ≥10 microns (black bars) and ≥25 microns (gray bars) after 3 months at 5°C and 25°C for F1, F2, F3, F4, F5, F9, F12, and F17. (b) Summary of particle count (particles / mL) for all size ranges: 2-5 microns (black bars), 5-10 microns (gray bars), 10-25 microns (horizontal stripes), and 10-25 microns (diagonal stripes) after 3 months at 5°C and 25°C for F1, F2, F3, F4, F5, F9, F12, and F17. [Figure 8] This figure shows the results at the start (T0) for % acidic and % basic species, measured by cation exchange chromatography. [Figure 9] This figure shows a typical chromatogram obtained from RP-HPLC to quantify the oxidation of Fc polymer molecules (CSL730) in a pharmaceutical formulation. [Figure 10] This figure shows plots comparing the initial (T0) % short-chain oxidized species, long-chain oxidized species, and relative % total oxidation for all formulations of the Fc polymer molecule (CSL730), as measured by RP-HPLC. [Figure 11] This figure shows plots of the % total oxidation progress, measured by RP-HPLC, for all formulations of the Fc polymer molecule (CSL730) at 25°C and 40°C during the 2-month storage stability period. [Figure 12]This figure shows an Fc construct containing three Fc domains formed from four polypeptide-like CSL730s. The first polypeptide (502) contains one Fc polypeptide containing amino acids of different charges at the CH3-CH3 interface, in addition to a wt sequence (506) joined in series with a protrusion-containing Fc polypeptide (504). The second polypeptide (508) contains an Fc polypeptide containing amino acids of different charges at the CH3-CH3 interface, in addition to a wt sequence (512) joined in series with another protrusion-containing Fc polypeptide (510). The third and fourth polypeptides (514 and 516, respectively) each contain a recess-containing Fc polypeptide. [Modes for carrying out the invention]

[0017] In the first aspect, the present invention is a) Fc polymers with concentrations between 60 mg / ml and 180 mg / ml b) pH values ​​between 4.8 and 6.0, and c) Stabilizers at concentrations between 200 mM and 450 mM This relates to an Fc polymer composition containing the above.

[0018] The results of the examples demonstrate that the Fc polymer composition described in the present invention is particularly stable. In particular, the examples show that, using the conditions described in the claims, even Fc polymer compositions with higher protein concentrations are prevented from protein precipitation and amide degradation, resulting in a reduction in the fragmentation rate.

[0019] The term "Fc polymer" as used in this invention refers to a polymer composed of at least two Fc moieties.

[0020] The terms “Fc moiety,” “Fc monomer,” “Fc fragment,” or “Fc domain,” as used interchangeably herein, describe a molecule assembled from two Fc polypeptides capable of binding to an Fc receptor.

[0021] The term "Fc polypeptide" as used in this invention refers to a polypeptide chain consisting of the heavy chain constant domain portion of an antibody.

[0022] In particular, the Fc polypeptide of the present invention comprises an antibody constant domain (CH2 and / or CH3) or functional fragments thereof (e.g., (i) a fragment that can dimerize with another Fc polypeptide to form an Fc moiety, and (ii) a fragment that can bind to an Fc receptor).

[0023] Furthermore, the Fc portion may include a hinge region and the constant CH2 and CH3 (and CH4) domains of the immunoglobulin heavy chain constant domain, or any combination of these fragments.

[0024] The term "constant domain CH2 / CH3 / CH4" as used in this invention refers to the constant domain portion of the antibody's heavy chain.

[0025] The term "hinge region" as used in this invention describes the mobile stretch of the polypeptide chain in the constant domain of the antibody heavy chain, whose usual function is to connect the Fab and Fc portions of the antibody, or fragments thereof.

[0026] In particular, the Fc polypeptide of the present invention comprises or consists of a full-length or partial hinge region and antibody constant domains CH2 and / or CH3 and / or CH4. More particularly, the Fc polypeptide of the present invention comprises or consists of a hinge region and a second and / or third antibody constant domain (CH2 and / or CH3), preferably a hinge region and a second and third antibody constant domain (CH2 and CH3).

[0027] The Fc polypeptide of the present invention may be derived from IgA, IgD, IgG, IgE, or IgM antibodies, and in particular, the Fc polypeptide of the present invention is derived from an IgG antibody, preferably a human IgG antibody, and more particularly, the Fc polypeptide of the present invention may be derived from one of the IgG antibody subclasses IgG1, IgG2a, IgG2b, IgG3, or IgG4. Most preferably, the Fc polypeptide of the present invention may be derived from human IgG1.

[0028] Typically, the Fc polypeptide described in the present invention does not contain any portion of the immunoglobulin that can act as an antigen recognition region or any complementarity-determining region (CDR).

[0029] The Fc polypeptide may be in its wild-type form, or it may contain one or more changes compared to the wild-type Fc polypeptide sequence.

[0030] Such changes may be any known changes to amino acids known to those skilled in the art, such as amino acid substitutions, additions, or deletions. Furthermore, the changes may be conservative or any changes that alter the interaction between two Fc polypeptides, between an Fc polypeptide or Fc domain and an Fc receptor, or between an Fc polypeptide or Fc domain and the complement system.

[0031] The alteration is introduced at any location in the Fc polypeptide, particularly in the hinge region, CH2, or CH3 domain.

[0032] Preferred modifications are known to those skilled in the art. In particular, the modifications may include knob-into-hole amino acid modifications, amino acid modifications resulting in disulfide bonds, amino acid modifications resulting in charged amino acid interactions, and the incorporation of peptide linkers that improve the interaction of Fc polypeptides at the Fc moiety.

[0033] The use of engineered recesses and engineered protrusions (or the “knob into hole” strategy) is well known to those skilled in the art and is described, for example, by Carter and collaborators (Ridgway et al., Protein Eng. 9: pp. 617-612, 1996; Atwell et al., J Mol Biol. 270: pp. 26-35, 1997; Merchant et al., Nat Biotechnol. 16: pp. 677-681, 1998), and is also disclosed in U.S. Patent No. 5,731,168.

[0034] In this invention, the engineered recesses and engineered protrusions are used in the manufacture of the Fc portion described herein.

[0035] The term "engineered recess" refers to the substitution of at least one original amino acid residue in a CH3 constant domain with a different amino acid residue having a smaller side chain volume than the original amino acid residue, thus creating a three-dimensional recess in the CH3 constant domain, for example.

[0036] As used herein, the term “engineered protrusion” refers to the substitution of at least one original amino acid residue in a CH3 constant domain, for example, with a different amino acid residue having a larger side chain volume than the original amino acid residue, and thus creating a three-dimensional protrusion in a CH3 constant domain.

[0037] Engineering-fabricated recesses are constructed by replacing amino acids containing large side chains, such as tyrosine or tryptophan, with amino acids containing small side chains, such as alanine, valine, or threonine. Similarly, engineering-fabricated protrusions are constructed by replacing amino acids containing small side chains, such as alanine, valine, or threonine, with amino acids containing large side chains, such as tyrosine or tryptophan. Methods for amino acid substitution are well known to those skilled in the art.

[0038] In the Fc portion described in the present invention, two Fc polypeptides may interact, thereby the first Fc polypeptide may include an engineered recess in its CH3 constant domain, while the second Fc polypeptide may include an engineered protrusion in its CH3 constant domain.

[0039] In other embodiments, an engineered recess or projection of one CH3 constant domain is engineered to better fit the original amino acid of another CH3 constant domain.

[0040] In this invention, the engineering of disulfide bonds in the CH3 internal domain is further applied to enhance dimerization. For example, a non-cysteine ​​amino acid in one CH3 constant domain is replaced with a cysteine ​​amino acid from another Fc polypeptide, particularly a cysteine ​​amino acid, to enable disulfide bond formation by the CH3 constant domain of the Fc polypeptide.

[0041] Furthermore, dimerization can be enhanced by incorporating one or more peptide linkers. Such peptide linkers typically contain mobile amino acids such as glycine and / or serine. Thereafter, the first Fc polypeptide is linked to the second Fc polypeptide by the peptide linker, the N-terminus of the peptide linker is linked to the C-terminus of the first Fc polypeptide through a chemical bond, and the C-terminus of the peptide linker is linked to the N-terminus of the second Fc polypeptide through a chemical bond. Suitable peptide linkers are well known to those skilled in the art.

[0042] Furthermore, the two Fc polypeptides are linked by a glycine linker; that is, the two parts of the Fc polypeptide are linked via amino acid chains of two or more glycine molecules.

[0043] If the Fc polypeptide contains one or more changes, these may be at any position on the molecule.

[0044] The Fc portion described in this invention is typically assembled from two Fc polypeptides. In particular, the Fc portion described in this invention is not understood to be a whole antibody.

[0045] The Fc portion consists of two identical Fc polypeptides. Furthermore, the Fc portion consists of two Fc polypeptides that are different from each other.

[0046] In the Fc portion, both Fc polypeptides may be mutated as described above, both Fc polypeptides may be wild-type, or one Fc polypeptide may be mutated as described above and the other Fc polypeptide may be wild-type.

[0047] In the Fc portion, Fc polypeptides can interact via any known species of intermolecular and intramolecular forces known to those skilled in the art, such as covalent or non-covalent bonds. Fc polypeptides can interact, for example, via inversely charged amino acids, which are introduced into their amino acid sequences by amino acid substitution as described above. Furthermore, Fc polypeptides can interact via disulfide bonds derived from the coupling of two thiol groups from two cysteine ​​residues of different Fc polypeptides. In addition, Fc polypeptides can be linked via the peptide linkers described above.

[0048] In particular, an Fc multimer is composed of 2 to 10 Fc parts, more particularly, an Fc multimer is composed of 3 to 6 Fc parts, and most particularly, an Fc multimer is composed of 3 Fc parts.

[0049] In a preferred embodiment, the Fc polymer is composed of 3 to 6 Fc portions.

[0050] In another preferred embodiment, the Fc polymer consists of three Fc parts.

[0051] In some embodiments, the Fc polymer comprises four polypeptides forming three Fc moieties, the first polypeptide comprising a first Fc polypeptide, a first linker, and a second Fc polypeptide; the second polypeptide comprising a third Fc polypeptide, a second linker, and a fourth Fc polypeptide; the third polypeptide comprising a fifth Fc polypeptide; the fourth polypeptide comprising a sixth Fc polypeptide; the first and third Fc polypeptides forming the first Fc moiety; the fifth and second Fc polypeptides forming the second Fc moiety; and the sixth and fourth Fc polypeptides forming the third Fc moiety.

[0052] In some embodiments, the Fc polymer has an antigen recognition region, for example, a variable domain (e.g., V H , V L It does not contain a hypervariable region (HVR) or a complementarity-determining region (CDR).

[0053] In some embodiments of this model, each of the first and third Fc polypeptides includes a complementary dimerization selectivity module that promotes dimerization between the first and third Fc polypeptides; and / or each of the second and fifth Fc polypeptides includes a complementary dimerization selectivity module that promotes dimerization between the second and fifth Fc polypeptides; and / or each of the fourth and sixth Fc polypeptides includes a complementary dimerization selectivity module that promotes dimerization between the fourth and sixth Fc polypeptides.

[0054] In some embodiments, the complementary dimerization selectivity module facilitates the selective dimerization of Fc polypeptides. In any of the Fc constructs described herein, the Fc polypeptides may have different sequences between two Fc polypeptides (i.e., between the Fc polypeptide and another Fc peptide of the Fc construct), for example, 20 or fewer amino acids (e.g., 15, 10 or fewer amino acids), for example, 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 or fewer amino acids. For example, the complementary dimerization selectivity module of any Fc construct facilitates the selective dimerization of one Fc polypeptide. H 3. An engineered recess was created in the constant domain of the antibody, and the C of the other Fc polypeptide. H Since the Fc polypeptide sequence of the constructs described herein can vary, as it may include an engineered protrusion of the antibody constant domain, the engineered recess and engineered protrusion are positioned to form a protrusion-recess pair of the Fc polypeptide. In some embodiments, the Fc construct is C H The 3 domains include amino acid modifications. In some embodiments, the Fc construct is C of an Fc polypeptide (one or more Fc polypeptides) for selective dimerization. H The three domains include amino acid modifications. Exemplary engineering-created recesses and protrusions are known in the art. In other embodiments, the complementary dimerization selectivity module is C of one Fc polypeptide. H 3. Negatively charged amino acids, engineered (substituted) into the constant domain of the antibody, are added to the C of the other Fc polypeptide. H The antibody constant domain contains an engineered (substituted) positively charged amino acid, and the negatively charged and positively charged amino acids are positioned to promote Fc domain formation between complementary Fc polypeptides. Exemplary complementary amino acid changes are known in the art. In some embodiments, one or more Fc polypeptides have the same sequence. In some embodiments, one or more Fc polypeptides have the same modification. In some embodiments, one, two, three, or four Fc polypeptides have the same modification.

[0055] In some embodiments, the Fc multimer comprises at least two Fc monomers connected via a linker. In some embodiments, the Fc multimer comprises at least one linker. The linker may be an amino acid spacer comprising 3 to 200 amino acids (such as 3 to 150, 3 to 100, 3 to 60, 3 to 50, 3 to 40, 3 to 30, 3 to 20, 3 to 10, 3 to 8, 3 to 5, 4 to 30, 5 to 30, 6 to 30, 8 to 30, 10 to 20, 10 to 30, 12 to 30, 14 to 30, 20 to 30, 15 to 25, 15 to 30, 18 to 22, and 20 to 30 amino acids). Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In certain embodiments, the linker may contain a motif, such as a single, multiple or repeating motif of GS, GGS, GGSG, GGGGS, GGG, GGGG. In certain embodiments, the linker may comprise GS, GGS, GGSG, GGGGS, GGG, GGGG or any of SEQ ID NOs: 34-61. In some embodiments, a linker is used to connect two Fc polypeptides in series. In other embodiments, a linker is used to connect C L and C H1 antibody constant domains. In other embodiments, the linker can also contain amino acids other than glycine and serine.

[0056] In some embodiments described herein, the Fc multimer can comprise a polypeptide comprising SEQ ID NOs: 1-31 or SEQ ID NOs: 1-31 having a maximum of 10 (such as a maximum of 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (such as substitutions, such as conservative substitutions). In some embodiments, the Fc polypeptides of the Fc domain of the construct can have different sequences (such as 20 or fewer amino acids, such as 15, 10 or fewer amino acids, such as 20, 15, 10, 8, 7, 6, 5, 4, 3 or 2 or fewer amino acids having different sequences) between two Fc polypeptides (i.e., between the Fc polypeptide of the Fc construct and another Fc polypeptide).

[0057] In some embodiments, one or more polypeptides of the Fc construct contain terminal lysine residues. In some embodiments, one or more polypeptides of the Fc construct do not contain terminal lysine residues. In some embodiments, all Fc polypeptides of the Fc construct contain terminal lysine residues. In some embodiments, all Fc polypeptides of the Fc construct do not contain terminal lysine residues. In some embodiments, terminal lysine residues can be removed from Fc polypeptides consisting of, or essentially consisting of, any one of the sequences of SEQ ID NOs: 2, 4, 5, 7, 9, 11, 13, 15, 17, 19, and 21 to produce a corresponding Fc polypeptide that does not contain terminal lysine residues. In some embodiments, terminal lysine residues can be added to Fc polypeptides consisting of, or essentially consisting of, the sequences of SEQ ID NOs: 1, 3, 6, 8, 10, 12, 14, 16, 18, 20, and 22-31 to produce a corresponding Fc polypeptide that contains terminal lysine residues.

[0058] In one embodiment, the Fc polymer of the present invention is disclosed in WO2015 / 168643A2, WO2017 / 205436A1, WO2017 / 205434A1, and WO2018 / 129255A1, which are incorporated herein by reference in their entirety. Preferably, the Fc polymer comprises an Fc construct having 2 to 10 Fc domains, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains. In some embodiments, the Fc polymer comprises 3 Fc domains.

[0059] In one embodiment, the disclosure features an Fc construct comprising four polypeptides forming three Fc domains. The first polypeptide has the formula ALB, where A comprises the first Fc polypeptide; L is a linker; and B comprises the second Fc polypeptide. The second polypeptide has the formula A'-L'-B', where A' comprises the third Fc polypeptide; L' is a linker; and B' comprises the fourth Fc polypeptide. The third polypeptide comprises the fifth Fc polypeptide, and the fourth polypeptide comprises the sixth Fc polypeptide. In this embodiment, A and A' combine to form the first Fc domain, B and the fifth Fc polypeptide combine to form the second Fc domain, and B' and the sixth Fc polypeptide combine to form the third Fc domain.

[0060] In some embodiments of this model, A and A' each include a dimerization selectivity module that promotes dimerization between these Fc polypeptides. In other embodiments, B and the fifth Fc polypeptide each include a dimerization selectivity module that promotes dimerization between these Fc polypeptides. In yet another embodiment, B' and the sixth Fc polypeptide each include a dimerization selectivity module that promotes dimerization between these Fc polypeptides.

[0061] In some embodiments of this model, one or more of A, B, A', B', a third polypeptide, and a fourth polypeptide are Fc polypeptides.

[0062] In some embodiments of this model, B and B' each contain mutations D399K and K409D, A and A' each contain mutations S354C, T366W, and E357K, and the fifth and sixth Fc polypeptides each contain mutations Y349C, T366S, L368A, Y407V, and K370D. Numbering refers to the Kabat EU numbering system for IgG, which is well known to those skilled in the art throughout this document.

[0063] In some embodiments of this model, A and A' each contain mutations D399K and K409D, B and B' each contain mutations S354C, T366W, and E357K, and the fifth and sixth Fc polypeptides each contain mutations Y349C, T366S, L368A, Y407V, and K370D.

[0064] In some embodiments of this model, each of L and L' contains at least 4, 8, 12, 14, 16, 18, or 20 glycine molecules. In some embodiments, each of L and L' contains 4 to 30, 8 to 30, or 12 to 30 glycine molecules. In some embodiments of this model, each of L and L' contains, consists of, or is fundamentally based on GGGGGGGGGGGGGGGGGGGG (Sequence ID 61).

[0065] In some embodiments, the Fc construct further comprises a heterogeneous portion, such as a peptide, for example, an albumin-conjugated peptide linked to the N-terminus or C-terminus of B or B' by a linker.

[0066] In other embodiments, the first and second polypeptides of the Fc construct have the same amino acid sequence, and the third and fourth polypeptides of the Fc construct have the same amino acid sequence.

[0067] In some embodiments, the first and second polypeptides include, consist of, or are based on, the sequence of SEQ ID NO: 24, and the third and fourth polypeptides include, consist of, or are based on, the sequence of SEQ ID NO: 23. In some embodiments, the Fc polymer in the composition includes two polypeptides having the amino acid sequence of SEQ ID NO: 23 and two polypeptides having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc polymer in the composition includes two polypeptides having the amino acid sequence of SEQ ID NO: 23 and two polypeptides having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc polymer in the composition comprises two polypeptides having the amino acid sequence of SEQ ID NO: 24 and two polypeptides having the amino acid sequence of SEQ ID NO: 23, with about 12% of the polypeptides having an asparagine-to-glutamine substitution at position 162. In some embodiments of the present disclosure, each of the first and second polypeptides comprises, or basically comprises, the sequence of SEQ ID NO: 24 having up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and the third and fourth polypeptides comprise, or basically comprise, the sequence of SEQ ID NO: 23 having up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions). In some cases, the first and second polypeptides contain, consist of, or essentially consist of the sequence of sequence number 29, and the third and fourth polypeptides contain, consist of, or essentially consist of the sequence of sequence number 28.In some embodiments of the present disclosure, each of the first and second polypeptides comprises, or essentially comprises, the sequence of SEQ ID NO: 29 having up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions), and the third and fourth polypeptides comprise, or essentially comprise, the sequence of SEQ ID NO: 28 having up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid modifications (e.g., substitutions, e.g., conservative substitutions).

[0068] In some embodiments of this aspect of the present disclosure, each of the first and third Fc polypeptides includes a complementary dimerization selectivity module that facilitates dimerization between the first and third Fc polypeptides, and each of the second and fourth Fc polypeptides includes a complementary dimerization selectivity module that facilitates dimerization between the second and fourth Fc polypeptides. In some embodiments, each of the complementary dimerization selectivity modules of the first and second Fc polypeptides includes an engineered projection, and each of the complementary dimerization selectivity modules of the third and fourth Fc polypeptides includes an engineered recess.

[0069] In some embodiments, one or more Fc domain monomers are IgG hinge regions or portions thereof, IgG C H 2 antibody constant domains, and IgG C H It contains 3 antibody constant domains. In some embodiments, each of the Fc polypeptides is an IgG hinge region or a portion thereof, IgG C H 2 antibody constant domains, and IgG C H It contains three antibody constant domains. In some embodiments, each of the Fc polypeptides is an IgG1 Fc polypeptide, preferably a human IgG1 Fc polypeptide.

[0070] In some embodiments of the two preceding aspects of this disclosure, one or more N-terminal aspartates of the first, second, third, and fourth polypeptides are mutated to glutamine. In some embodiments, the N-terminal aspartates of each of the first, second, third, and fourth polypeptides are mutated to glutamine.

[0071] In some embodiments, one or more of the first, second, third, and fourth polypeptides lack C-terminal lysine.

[0072] In some embodiments, the first polypeptide and the second polypeptide have the same amino acid sequence, and the third polypeptide and the fourth polypeptide have the same amino acid sequence. In some embodiments, the first polypeptide and the second polypeptide do not have the same amino acid sequence. In some embodiments, the third polypeptide and the fourth polypeptide do not have the same amino acid sequence.

[0073] In some embodiments, at least one Fc domain includes an amino acid modification that alters one or more of the following: (i) binding affinity to one or more Fc receptors, (ii) effector function, (iii) level of Fc domain sulfated, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and / or (vii) sensitivity to degradation. In some embodiments, the Fc domain includes an amino acid modification that alters binding affinity to one or more Fc receptors, e.g., S267E / L328F. In some embodiments, the Fc receptor is FcγRIIb. In some cases, the modifications described herein increase affinity to the FcγRIIb receptor. In some cases, the S267E / L328F modification increases binding affinity to FcγRIIb. In some embodiments, the Fc domain includes an amino acid modification that alters the level of Fc domain sulfated, e.g., 241F, 243F, 246K, 260T, or 301R. In some embodiments, the Fc domain includes amino acid modifications that alter protease resistance, selected from, for example, the following sets: 233P, 234V, 235A, and 236del; 237A, 239D, and 332E; 237D, 239D, and 332E; 237P, 239D, and 332E; 237Q, 239D, and 332E; 237S, 239D, and 332E; 239D, 268F, 324T, and 332E; 239D, 326A, and 333A; 239D and 332E; 243L, 292P, and 300L; 267E, 268F, 324T, and 332E; 267E and 332E; 268F, 324T, and 332E; 326A, 332E, and 333A; or 326A and 333A. In some embodiments, the Fc domain includes amino acid modifications that alter the sensitivity of the Fc domain to degradation, e.g., C233X, D234X, K235X, S236X, T236X, H237X, C239X, S241X, and G249X, where X is any amino acid.

[0074] In some embodiments, the Disclosure features an Fc construct comprising a)i) a first Fc polypeptide; ii) a second Fc polypeptide; and iii) a first polypeptide comprising a linker connecting the first Fc polypeptide to the second Fc polypeptide; b)i) a third Fc polypeptide; ii) a fourth Fc polypeptide; and iii) a second polypeptide comprising a linker connecting the third Fc polypeptide to the fourth Fc polypeptide; c) a third polypeptide comprising a fifth Fc polypeptide; and d) a fourth polypeptide comprising a sixth Fc polypeptide, wherein the first and fifth Fc polypeptides combine to form a first Fc domain, the second and fourth Fc polypeptides combine to form a second Fc domain, and the third and sixth Fc polypeptides combine to form a third Fc domain, and at least one Fc domain comprises an amino acid modification at position I253 (e.g., a single amino acid modification at position I253).

[0075] In some embodiments, the first and second polypeptides are identical to each other, and the third and fourth polypeptides are identical to each other. In some embodiments, the first Fc domain contains an amino acid modification at position I253. In some cases, one or both of the first and fifth Fc polypeptides contain an amino acid substitution at position I253. In some embodiments, the second Fc domain contains an amino acid modification at position I253. In some embodiments, one or both of the second and fourth Fc polypeptides contain an amino acid substitution at position I253. In some embodiments, the third Fc domain contains an amino acid modification at position I253. In some embodiments, one or both of the third and sixth Fc polypeptides contain an amino acid substitution at position I253. In some embodiments, each amino acid modification (e.g., substitution) at position I253 is independently selected from the group consisting of I253A, I253C, I253D, I253E, I253F, I253G, I253H, I253I, I253K, I253L, I253M, I253N, I253P, I253Q, I253R, I253S, I253T, I253V, I253W, and I253Y. In some embodiments, each amino acid modification (e.g., substitution) at position I253 is I253A.

[0076] In another embodiment, the present disclosure features an Fc construct comprising a)i) a first Fc polypeptide; ii) a second Fc polypeptide; and iii) a first polypeptide comprising a linker connecting the first Fc polypeptide to the second Fc polypeptide; b)i) a third Fc polypeptide; ii) a fourth Fc polypeptide; and iii) a second polypeptide comprising a linker connecting the third Fc polypeptide to the fourth Fc polypeptide; c) a third polypeptide comprising a fifth Fc polypeptide; and d) a fourth polypeptide comprising a sixth Fc polypeptide, wherein the first and fifth Fc polypeptides combine to form a first Fc domain, the second and fourth Fc polypeptides combine to form a second Fc domain, and the third and sixth Fc polypeptides combine to form a third Fc domain, and at least one Fc domain comprising an amino acid modification at the R292 position (e.g., a single amino acid modification).

[0077] In some embodiments, the first Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the first and fifth Fc polypeptides contain an amino acid substitution at position R292. In some embodiments, the second Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the second and fourth Fc polypeptides contain an amino acid substitution at position R292. In some embodiments, the third Fc domain contains an amino acid modification at position R292. In some embodiments, one or both of the third and sixth Fc polypeptides contain an amino acid substitution at position R292. In some embodiments, each of the first, second, and third Fc domains contains an amino acid modification (e.g., substitution) at position R292. In some embodiments, each of the first, second, and third Fc domains contains the amino acid modification (e.g., substitution) R292P (i.e., each Fc monomer has the R292P modification compared to, for example, SEQ ID NO: 23). In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0078] In some embodiments, each amino acid modification (e.g., substitution) at position R292 is independently selected from R292D, R292E, R292L, R292P, R292Q, R292R, R292T, or R292Y. In some embodiments, each amino acid modification (e.g., substitution) at position R292 is R292P. In some embodiments, each of the first and third Fc domains contains the amino acid modification (e.g., substitution) I253A, and each of the first, second, and third Fc domains contains the amino acid modification (e.g., substitution) R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A, one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, each of the first, second, and third Fc domains contains the amino acid modifications (e.g., substitutions) I253A and R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A, one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0079] In some embodiments, the first Fc domain and the third Fc domain each contain amino acid substitutions I253A and R292P, and the second Fc domain contains amino acid substitution R292P. In some cases, one or both of the first and fifth Fc polypeptides contain amino acid substitution I253A; one or both of the first and fifth Fc polypeptides contain amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain amino acid substitution I253A; one or both of the third and sixth Fc polypeptides contain amino acid substitution R292P; and one or both of the second and fourth Fc polypeptides contain amino acid substitution R292P.

[0080] In some embodiments, the second Fc domain contains the amino acid substitution I253A. In some embodiments, one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A. In some embodiments, each of the first and third Fc domains contains the amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A. In some embodiments, each of the first, second, and third Fc domains contains the amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A, one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A.

[0081] In some embodiments, the second Fc domain contains the amino acid substitution R292P. In some embodiments, one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, the second Fc domain contains the amino acid substitutions I253A and R292P. In some embodiments, one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A, and one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, each of the first and third Fc domains contains the amino acid substitution I253A, and the second Fc domain contains the amino acid substitution R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A; one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A; and one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P.

[0082] In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitution I253A, and the second Fc domain contains the amino acid substitution I253A and R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A; one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A; one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A; and one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitution R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0083] In some embodiments, the first and third Fc domains contain the amino acid substitution R292P, and the second Fc domain contains the amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P; and one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A. In some embodiments, each of the first and third Fc domains contains I253A and R292P (e.g., the amino acid substitutions I253A and R292P). In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A; one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A; and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0084] In some embodiments, the first Fc domain and the third Fc domain each contain amino acid substitutions I253A and R292P, and the second Fc domain contains amino acid substitution I253A. In some embodiments, one or both of the first and fifth Fc polypeptides contain amino acid substitution I253A; one or both of the first and fifth Fc polypeptides contain amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain amino acid substitution I253A; one or both of the third and sixth Fc polypeptides contain amino acid substitution R292P; and one or both of the second and fourth Fc polypeptides contain amino acid substitution I253A. In some embodiments, the first Fc domain, the second Fc domain, and the third Fc domain each contain amino acid substitution R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P; one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P; and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0085] In some embodiments, the first Fc domain and the third Fc domain each contain the amino acid substitution R292P, and the second Fc domain contains the amino acid substitutions I253A and R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P; one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A; and one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, the first Fc domain, the second Fc domain, and the third Fc domain each contain the amino acid substitutions I253A and R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution I253A; one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P; one or both of the second and fourth Fc polypeptides contain the amino acid substitution I253A; one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P; one or both of the third and sixth Fc polypeptides contain the amino acid substitution I253A; and one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P.

[0086] In some embodiments, each of the first, second, and third Fc domains contains the amino acid substitution R292P. In some embodiments, one or both of the first and fifth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, one or both of the third and sixth Fc polypeptides contain the amino acid substitution R292P. In some embodiments, one or both of the second and fourth Fc polypeptides contain the amino acid substitution R292P.

[0087] In some embodiments, the Fc constructs described herein do not include an antigen recognition region, such as a variable domain or complementarity-determining region (CDR). In some embodiments, the Fc construct (or Fc domain within the Fc construct) is formed in whole or in part by the association of Fc polypeptides present in different polypeptides. In certain embodiments, the Fc construct does not include any additional domains (e.g., an IgM tail or an IgA tail) that facilitate the association of the two polypeptides. In other embodiments, covalent bonds (e.g., disulfide crosslinks) are present only between two Fc polypeptides that conjugate to form an Fc domain. In other embodiments, the Fc construct does not include covalent bonds (e.g., disulfide crosslinks) between Fc domains. In yet another embodiment, the Fc construct provides sufficient structural mobility so that all or substantially all of the Fc domains of the Fc construct can interact simultaneously with Fc receptors on the cell surface. In one embodiment, the Fc polypeptides differ in their primary sequences, either wild-type derived or derived from each other and having a dimerization-selective module.

[0088] In another aspect, the Disclosure features compositions and methods for promoting the selective dimerization of Fc polypeptides. The Disclosure comprises an Fc domain, wherein two Fc polypeptides of the Fc domain are C H This disclosure includes identical mutations at at least two positions within the ring of charged residues at the interface between the three antibody constant domains. H The method for creating such an Fc domain also includes the step of introducing complementary dimerization selectivity modules having identical mutations into two Fc polypeptide sequences at at least two positions within a ring of charged residues at the interface between three antibody constant domains. H The interface between the three antibody constant domains consists of a hydrophobic patch surrounded by a ring of charged residues. H 3. The antibody constant domain is a different C HWhen combined with the antibody constant domain, these charged residues pair with residues of the opposite charge. By reversing the charge of both members of two or more complementary residue pairs, mutant Fc polypeptides maintain complementarity with Fc polypeptides of the same mutant sequence but have lower complementarity with Fc polypeptides without those mutations. In this embodiment, the same dimerization selectivity module promotes homodimerization. Such Fc domains include Fc polypeptides containing the double mutations K409D / D399K, K392D / D399K, E357K / K370E, D356K / K439D, K409E / D399K, K392E / D399K, E357K / K370D, or D356K / K439E. In another embodiment, the Fc domain includes an Fc polypeptide containing a quadruple mutation, such as K409D / D399K / E357K / K370E, which is a combination of any pair of double mutations.

[0089] In another embodiment, in addition to identical dimerization selectivity modules, the Fc polypeptide of the Fc domain includes a complementary dimerization selectivity module with non-identical mutations that promotes a specific association (e.g., an engineered recess and protrusion). As a result, the two Fc polypeptides contain two dimerization selectivity modules, maintaining complementarity with each other but reduced complementarity with other Fc polypeptides. This embodiment promotes heterodimerization between an Fc polypeptide containing a recess and an Fc polypeptide containing a protrusion. In one example, complementary dimerization selectivity modules with non-identical mutations in the charged pair residues of both Fc polypeptides are combined with the protrusion of one Fc polypeptide and the recess of the other Fc polypeptide. In another embodiment, the Fc polypeptide of the Fc domain includes a complementary dimerization selectivity module with non-identical mutations that promote a specific association (e.g., an engineered recess and protrusion), but does not include an identical dimerization selectivity module.

[0090] In any Fc construct described herein, it is understood that the order of the Fc polypeptides is interchangeable. For example, in a polypeptide having the formula ALB, the carboxyl terminus of A is joined to the amino terminus of L, and then its carboxyl terminus is joined to the amino terminus of B. Alternatively, the carboxyl terminus of B is joined to the amino terminus of L, and then its carboxyl terminus is joined to the amino terminus of C. Both of these structures are encompassed by the formula ALB.

[0091] The properties of these constructs enable the efficient production of substantially homogeneous compositions. The degree of homogeneity of the composition affects the pharmacokinetics and the in vivo performance of the composition. Such homogeneity of the composition is desirable to ensure the safety, efficacy, uniformity, and reliability of the composition. The Fc constructs of this disclosure may be substantially homogeneous groups or compositions (e.g., at least 85%, 90%, 95%, 98%, or 99% homogeneity).

[0092] As described in further detail herein, the disclosure features substantially homogeneous compositions containing Fc constructs, all having the same number of Fc domains, and methods for preparing such substantially homogeneous compositions.

[0093] The Fc constructs of this disclosure can be included in a substantially homogeneous population of Fc constructs having 2 to 10 Fc domains (e.g., 2 to 8 Fc domains, 2 to 6 Fc domains, 2 to 4 Fc domains, 2 to 3 Fc domains, 3 to 5 Fc domains, or 5 to 10 Fc domains) (e.g., at least 85%, 90%, 95%, 98%, or 99% homogeneity), such as constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains, as described herein. As a result, it is possible to produce a pharmaceutical composition that does not have substantial aggregates or unwanted polymerization of the Fc constructs.

[0094] Furthermore, the Fc multimer is composed of six Fc moieties, these Fc moieties being IgG Fc moieties, and the addition of an 18-amino acid tail to the C-terminus of IgM results in hexamerization of IgG. A preferred example of such a hexamer is disclosed in WO2017 / 129737A1. For example, the Fc multimer contains six IgG Fc domains. Each IgG Fc domain contains two Fc polypeptides, each Fc polypeptide containing an IgG Fc polypeptide and an IgM tail.

[0095] In preferred embodiments, the Fc polypeptide further comprises an IgG hinge region, and the Fc polypeptide does not contain the Fab polypeptide.

[0096] For example, in one embodiment, the Fc polypeptide includes an IgG1 hinge region, an IgG1 Fc region, and an IgM tail fragment, but does not include a Fab polypeptide. In a preferred embodiment, the Fc polypeptide is SEQ ID NO: 32 and has up to five conserved amino acid changes.

[0097] In a preferred embodiment, the Fc polypeptide comprises an IgG1 hinge region, an IgG1 Fc region, and an IgM tail fragment, wherein the IgG1 Fc region has cysteine ​​instead of leucine at position 309 (according to EU numbering), the Fc polypeptide does not contain a Fab polypeptide, and the Fc polypeptide is Sequence ID No. 33. In one embodiment, the Fc polypeptide is Sequence ID No. 33 having up to five conserved amino acid changes.

[0098] In general, Fc polymers are composed of identical Fc moieties. Furthermore, Fc polymers are composed of Fc moieties that are all different from each other, partially identical, and partially different from each other in any combination.

[0099] In Fc polymers, all Fc portions may be mutated, or no Fc portions may be mutated, or any number of Fc portions may be mutated. In particular, the Fc portions described in this invention are not understood to be the whole antibody.

[0100] Fc polypeptides, Fc moieties, or Fc polymers are produced by any method of antibody production, Fc moiety production, or protein production known to those skilled in the art. Methods for producing Fc polypeptides, Fc moieties, or Fc polymers are generally well known to those skilled in the art. For example, Fc polypeptides, Fc moieties, or Fc polymers are produced with the help of mammalian cells, bacterial cells, or insect cells after introducing a protein-encoding nucleic acid into the mammalian cells, bacterial cells, or insect cells. Subsequently, the Fc polypeptides, Fc moieties, or Fc polymers are isolated from the mammalian cells, bacterial cells, or insect cells or recovered from the supernatant.

[0101] The Fc polymer composition described in the present invention contains an Fc polymer at a concentration between 60 mg / ml and 180 mg / ml.

[0102] The Fc polymer composition described in the present invention contains an Fc polymer at a concentration between 70 mg / ml and 160 mg / ml, more particularly at a concentration between 80 mg / ml and 120 mg / ml, even more particularly at a concentration between 90 mg / ml and 110 mg / ml, and most particularly at a concentration between 95 mg / ml and 105 mg / ml.

[0103] Furthermore, the Fc polymer composition described in the present invention contains an Fc polymer in a concentration between 70 mg / ml and 160 mg / ml, more particularly in a concentration between 70 mg / ml and 140 mg / ml, even more particularly in a concentration between 70 mg / ml and 120 mg / ml, and most particularly in a concentration between 70 mg / ml and 110 mg / ml.

[0104] In a preferred embodiment, the Fc polymer composition described in the present invention contains Fc polymers at concentrations of 70 mg / ml and 110 mg / ml.

[0105] Furthermore, the Fc polymer composition described in the present invention contains Fc polymers in particular at concentrations of 90 mg / ml and 180 mg / ml, more particularly at concentrations between 110 mg / ml and 180 mg / ml, even more particularly at concentrations between 115 mg / ml and 160 mg / ml, and most particularly at concentrations between 120 mg / ml and 160 mg / ml.

[0106] In another preferred embodiment, the Fc polymer composition described in the present invention contains the Fc polymer at a concentration between 110 mg / ml and 180 mg / ml, particularly at 120 mg / ml or 160 mg / ml.

[0107] The protein concentration of the Fc polymer composition described in the present invention is measured by preferred techniques known to those skilled in the art. For example, the protein concentration of the Fc polymer composition described in the present invention is measured by absorption spectroscopy, colorimetric analysis (e.g., Bradford method, Biuret method, bicinchoninic acid assay (BCA assay), or Lowry method), gravimetric analysis (e.g., Kjeldahl method), quantitative amino acid analysis (e.g., total hydrolysis, end group analysis, and Edman degradation), or refractive index measurement. Preferably, UV spectroscopy measuring absorption at 280 nm has been used to measure the protein concentration of the Fc polymer composition of the present invention.

[0108] The Fc polymer composition described in the present invention further comprises a pH value between 4.8 and 6.0.

[0109] In particular, the Fc polymer composition described in the present invention comprises a pH value between 4.9 and 5.8, more particularly, the Fc polymer composition described in the present invention comprises a pH value between 5.0 and 5.6, and most particularly, the Fc polymer composition described in the present invention comprises a pH value between 5.0 and 5.5.

[0110] In a preferred embodiment, the Fc polymer composition described in the present invention contains a pH value between 5.0 and 5.5.

[0111] The pH value of the Fc polymer composition of the present invention can be detected by any technique known to those skilled in the art for measuring pH values, such as a pH meter.

[0112] The Fc polymer composition described in the present invention further contains a stabilizer at a concentration between 200 mM and 450 mM.

[0113] The term "stabilizer" as used in this invention may refer to any substance that prevents the decomposition of any component of the composition of the present invention. In particular, the stabilizer is a substance that prevents the decomposition and / or aggregation of the Fc polymer and / or Fc portion of the composition of the present invention.

[0114] Suitable stabilizers are well known to those skilled in the art. For example, suitable stabilizers may be any antioxidant (e.g., to prevent auto-oxidation), any scavenger (e.g., to deactivate metal ions that may act as catalysts), or any UV stabilizer (e.g., to prevent decomposition by UV irradiation).

[0115] In particular, the stabilizers used in the present invention may be polyols and / or amino acids.

[0116] The polyol used as a stabilizer in the composition of the present invention may be an organic molecule based on 2 to 20 carbon atoms, particularly 3 to 18 carbon atoms, more particularly 4 to 15 carbon atoms, and most particularly 5 to 12 carbon atoms. Furthermore, the polyol may be linear or branched. Furthermore, the polyol may be a cyclic molecule containing one, two or more rings. The polyol may contain at least two, three, four, five, six, seven, or eight hydroxyl groups. The polyol may further be aliphatic, alicyclic, or aromatic.

[0117] Suitable polyols to be used as stabilizers in the compositions of the present invention are well known to those skilled in the art and may be disaccharides, glycerol, mannitol, inositol, xylitol, erythritol and / or adonitol. More particularly, the stabilizers are disaccharides such as sucrose or trehalose and / or sorbitol.

[0118] Suitable amino acids for use as stabilizers in the compositions of the present invention are well known to those skilled in the art. In particular, natural amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and / or valine may be used as stabilizers in the compositions of the present invention. More particularly, proline or arginine may be used as stabilizers in the compositions of the present invention.

[0119] In preferred embodiments, the Fc polymer composition described in the present invention contains a polyol, particularly a disaccharide, preferably sucrose or trehalose, or a reducing sugar such as sorbitol, as a stabilizer.

[0120] The compositions described in the present invention may contain one stabilizer, or a mixture of two, three, four, five or more stabilizers.

[0121] The compositions described in the present invention may contain a stabilizer in a concentration of 50 mM to 500 mM, more particularly a stabilizer in a concentration of 100 mM to 400 mM, more particularly a stabilizer in a concentration of 200 mM to 300 mM, and most particularly a stabilizer in a concentration of 225 mM to 275 mM.

[0122] The compositions described in the present invention may contain at least 200 mM sucrose, particularly 200 mM to 1000 mM sucrose, more particularly 200 mM to 750 mM sucrose, and most particularly 220 mM to 500 mM sucrose. When sucrose is used in combination with one or more other stabilizers, lower concentrations, such as at least 100 mM sucrose or 100 mM to 250 mM sucrose, may be used.

[0123] The compositions described in the present invention may contain 100 mM to 500 mM trehalose, particularly 150 mM to 400 mM trehalose, and more particularly 200 mM to 300 mM trehalose.

[0124] The compositions described in the present invention may contain 50 mM to 500 mM sorbitol, particularly 100 mM to 400 mM sorbitol, more particularly 200 mM to 300 mM sorbitol, and most particularly 225 mM to 275 mM sorbitol.

[0125] In the Fc polymer composition described in the present invention, the molar ratio of the Fc polymer to the stabilizer may be between 1:100 and 1:800, particularly between 1:300 and 1:650, more particularly between 1:350 and 1:500, and most particularly between 1:400 and 1:425.

[0126] In preferred embodiments, the molar ratio of the Fc polymer to the stabilizer in the Fc polymer composition described in the present invention is between 1:300 and 1:650, particularly between 1:400 and 1:425. Preferably, the Fc polymer composition of this embodiment contains 10% or less, more preferably 7% or less, even more preferably 5% or less, and most preferably 3.5% or less of aggregates (as determined by SEC) when stored at 2-8°C for 12 months. Preferably, the Fc polymer composition of this embodiment contains 15% or less, more preferably 10% or less, even more preferably 8% or less, and most preferably 6.5% or less of aggregates when stored at 25°C.

[0127] Furthermore, the composition described in the present invention may or may not contain a buffer.

[0128] In a preferred embodiment, the Fc polymer composition described in the present invention includes a buffer.

[0129] The term "buffer" as used in this invention refers to a mixture containing a weak acid and its conjugate base, or vice versa, which remains in its buffer region, maintaining its pH at approximately constant values ​​when further acids or bases are added thereto.

[0130] The Fc polymer composition of the present invention may contain one or more buffers. Suitable buffers for the present invention are well known to those skilled in the art. In general, any buffer suitable for pharmaceutical applications, and especially suitable for administration to the body of a subject such as a human patient, can be used. Examples of suitable buffers within the pH range required for the present invention are phosphate buffer, glutamate buffer, acetate buffer, histidine buffer, and / or citrate buffer. In particular, the buffer is acetate buffer, histidine buffer, or citrate buffer.

[0131] In a preferred embodiment, the Fc polymer composition described in the present invention comprises a buffer, the buffer being an acetate buffer, a histidine buffer, or a citrate buffer. In a preferred embodiment, the Fc polymer composition described in the present invention comprises a histidine buffer.

[0132] The compositions described in the present invention may further contain antioxidants. The term "antioxidant" as used in the present invention refers to a substance that inhibits oxidation. Preferably, it inhibits the oxidation of the Fc polymer or the Fc portion of the Fc polymer of the composition or the Fc polypeptide. Suitable antioxidants are well known to those skilled in the art. For example, any antioxidant suitable for pharmaceutical applications, particularly for administration to the body of a subject such as a human patient, may be used. The Fc polymer compositions of the present invention may contain one or more antioxidants.

[0133] Examples of suitable antioxidants include ascorbic acid, vitamin E, reduced glutathione (GSH), and / or methionine. In particular, the Fc polymer composition described in the present invention may contain methionine.

[0134] The preferred amounts of antioxidants used in the present invention are well known to those skilled in the art and depend on the type of antioxidant. Generally, the Fc polymer composition may contain 0.01 mM to 100 mM of antioxidants, particularly 0.05 mM to 50 mM of antioxidants, more particularly 0.1 mM to 30 mM of antioxidants, and most particularly 0.5 mM to 25 mM of antioxidants or even 1 mM to 20 mM.

[0135] In particular, the Fc polymer composition may contain 0.01 mM to 100 mM methionine, especially 0.05 mM to 20 mM methionine, more particularly 0.1 mM to 17 mM methionine, even more particularly 0.5 mM to 15 mM methionine, and most particularly, 1 mM to 12 mM methionine.

[0136] In a preferred embodiment, the Fc polymer composition described in the present invention further comprises an antioxidant, particularly methionine.

[0137] The compositions described in the present invention may further contain surfactants. The term “surfactant” as used in the present invention refers to a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Thereafter, when dissolved in water, surfactants can reduce the advancing contact angle and help to replace the air phase at the surface with the liquid phase.

[0138] The surfactants used in the Fc polymer compositions described in the present invention are well known to those skilled in the art. In particular, any surfactant suitable for pharmaceutical applications, especially for administration to the body of a subject such as a human patient, can be used.

[0139] For example, a surfactant may be one of the subclasses of anionic surfactants, nonionic surfactants, or zwitterionic surfactants, or it may belong to the subclass of surfactants having a cationic head group.

[0140] In particular, nonionic surfactants are used. Suitable nonionic surfactants, such as polyoxyethylene glycol octylphenol ether (e.g., C8H17-(C6H4)-(O-C2H4)1-25-OH (e.g., Triton® X-100)), sorbitan alkyl esters (e.g., SPAN® and / or polyoxyethylene glycol sorbitan alkyl esters, e.g., polysorbate (e.g., polysorbate 20 or polysorbate 80)), and / or poloxamers (e.g., Antarox®) are well known to those skilled in the art. In particular, sorbitan alkyl esters, more particularly polyoxyethylene glycol sorbitan alkyl esters, even more particularly polysorbate, and most particularly polysorbate 80 are used.

[0141] Typically, anionic surfactants are also used. Suitable anionic surfactants, such as sodium dioctyl sulfosuccinate (DOSS), sodium lauryl sulfate ether (e.g., Taxapon®), ligninsulfonic acid, and / or sodium stearate, are well known to those skilled in the art.

[0142] In general, the compositions of the present invention may contain one or more surfactants. If a composition contains more than one surfactant, these may belong to the same or different subclasses of surfactants. Furthermore, if a composition contains more than one surfactant, these may be any mixture of the surfactants described above.

[0143] The preferred amounts of surfactants used in the present invention are well known to those skilled in the art and depend on the type of surfactant. Generally, the composition may contain 0.0001% w / v to 0.5% w / v of surfactant, particularly 0.001% w / v to 0.1% w / v of surfactant, more particularly 0.005% w / v to 0.05% w / v of surfactant, and most particularly 0.01% w / v to 0.03% w / v of surfactant. For example, the composition may contain 0.0001% w / v to 0.5% w / v of polysorbate 80, particularly 0.001% w / v to 0.1% w / v of polysorbate 80, more particularly 0.005% w / v to 0.05% w / v of polysorbate 80, and most particularly 0.01% w / v to 0.03% w / v of polysorbate 80.

[0144] In preferred embodiments, the Fc polymer composition described in the present invention further comprises a surfactant, particularly polysorbate 80.

[0145] The Fc polymer composition described in the present invention may further contain, but is not limited to, conventional pharmaceutical additives and adjuvants, excipients or diluents, such as water, salts, gelatin of any origin, vegetable gum, lignin sulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavorings, preservatives, emulsifiers, lubricants, colorants, wetting agents, fillers, etc., as well as other substances that do not react adversely with the active compound. The Fc polymer composition may further contain enzymes such as protease inhibitors.

[0146] In particular, the Fc polymer composition described in the present invention may further contain one or more salts. Suitable salts, such as sodium chloride, which are suitable for pharmaceutical applications, are well known to those skilled in the art. In particular, the Fc polymer composition described in the present invention may further contain salts in concentrations of 1 mM to 250 mM, more particularly salts in concentrations of 10 mM to 200 mM, and most particularly salts in concentrations of 30 mM to 170 mM.

[0147] In some cases, the suspension may also contain further suitable agents that increase the solubility of the compound, enabling the production of high-concentration solutions.

[0148] In one embodiment of the present invention, the Fc polymer composition described in the present invention may contain 70 mg / ml of Fc polymer, particularly CSL730, L-histidine buffer, sodium chloride and L-proline, polysorbate 80, and water for injection.

[0149] The Fc polymer composition is sterilized or treated by any method well known to those skilled in the art, making it suitable for storage.

[0150] In a second aspect, the present invention relates to an Fc polymer composition comprising the Fc polymer composition of the present invention in a freeze-dried form.

[0151] With respect to the Fc polymer composition described in the first embodiment, all of the above embodiments can also be applied to the second embodiment.

[0152] The term "freeze-drying" as used in this invention means any low-temperature dehydration method comprising the steps of freezing the product, reducing the pressure, and removing ice by sublimation.

[0153] The Fc polymer composition is freeze-dried by any freeze-drying technique known to those skilled in the art. Freeze-drying is carried out by any means and at any temperature. Preferably, freeze-drying is carried out at room temperature (RT).

[0154] The Fc polymer composition may be pretreated before freeze-drying, and may include, for example, a step of concentrating the Fc polymer composition.

[0155] Freeze-drying may involve a first stage of primary drying. During this primary drying stage, the pressure is reduced (to a range of a few millibars) to supply sufficient heat to the material for the ice to sublimate. The amount of heat required is calculated using the latent heat of sublimation of the sublimating molecules. In this initial drying stage, approximately 95% of the water in the material sublimes. This stage can be slow (it can take several days in industry) because excessive heat can alter the structure of the material.

[0156] In the primary drying stage, the pressure is controlled by applying a partial vacuum. Vacuum typically accelerates sublimation and is useful as an intentional drying method. Furthermore, a cooling condenser chamber and / or condenser plate can provide a surface for re-liquefying and solidifying water vapor.

[0157] Freeze-drying may include a second stage of secondary drying, in which the temperature is raised above the primary drying stage, and may even be above 0°C. Typically, the pressure is also reduced at this stage to facilitate desorption (typically in the range of microbars or fractions of pascals). However, some products may also benefit from increased pressure. After freeze-drying, buffer components may retain their dry salt form.

[0158] Furthermore, after the freeze-drying method, the substance is sealed in a glass vial or similar container. Generally, the resulting powder is reconstituted by adding a suitable solvent before use. The solution may be an aqueous solution such as buffered saline solution. Preferably, freeze-drying is carried out until (most) the liquid is removed. At the end of the freeze-drying method, the final residual water content in the product is usually extremely low. In particular, the final residual water content in the product is approximately 1% to 4%.

[0159] In particular, the lyophilized products described herein may be substantially pure and / or sterile.

[0160] In a preferred embodiment, the freeze-dried form of the Fc polymer composition of the present invention consists of 3 to 10 Fc portions.

[0161] In a more preferred embodiment, the freeze-dried form of the Fc polymer composition of the present invention is composed of three Fc portions.

[0162] In a third aspect, the present invention relates to an Fc polymer composition for use in the treatment of autoimmune or inflammatory diseases.

[0163] The term "autoimmune disease" as used in this invention describes a condition resulting from an abnormal immune response to a normal part of the body, and is a subtype of inflammatory disease.

[0164] Generally, the Fc multimer compositions of the present invention are used in the treatment of any autoimmune disease known to those skilled in the art. In particular, the Fc multimer compositions of the present invention are used in the treatment of any autoimmune disease known to those skilled in the art that can be treated by administration of an Fc moiety or an Fc multimer. Generally, the Fc multimer compositions of the present invention are used in the treatment of autoimmune diseases in any part of the body.

[0165] Examples of autoimmune diseases treated with the Fc multimer composition described in the present invention may include celiac disease, fulminant type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, idiopathic thrombocytopenic purpura (ITP), Kawasaki disease, Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), severe myasthenia gravis, ulcerative colitis, immune complex-mediated renal impairment, scleroderma, dermatomyositis, neuromyelitis optica, Sjögren's syndrome, systemic vasculitis, or systemic lupus erythematosus.

[0166] The term "inflammatory disease" as used in this invention describes a disorder or condition characterized by inflammation, i.e., a complex biological and defensive response of the body's cells and tissues to harmful stimuli (such as pathogens, damaged cells, or irritants). Inflammation typically involves immune cells, blood vessels, and molecular mediators.

[0167] Examples of inflammatory diseases include asthma, chronic peptic ulcers, tuberculosis, periodontitis, Crohn's disease, glomerulonephritis, graft rejection, sinusitis, or active hepatitis.

[0168] Furthermore, all of the above embodiments relating to the Fc polymer composition described in the first embodiment also apply to the third embodiment.

[0169] The use of the Fc multimer composition of the present invention in the treatment of autoimmune or inflammatory diseases may include any form of administration known to those skilled in the art, such as parenteral administration, subcutaneous, intravenous, and / or transdermal administration. In particular, the use of the Fc multimer composition of the present invention in the treatment of autoimmune or inflammatory diseases includes its subcutaneous administration.

[0170] Furthermore, the use of the Fc multimer composition of the present invention in the treatment of autoimmune or inflammatory diseases is accompanied by concomitant use of further immunoglobulins or a portion thereof, nonsteroidal anti-inflammatory drugs (NSAIDs), and / or immunosuppressants. The Fc multimer composition and further immunoglobulins or a portion thereof, nonsteroidal anti-inflammatory drugs (NSAIDs), and / or immunosuppressants are administered together in a single dose or separately in two or more doses.

[0171] The present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. Furthermore, the terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the invention. Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless otherwise explicitly indicated by the context. Similarly, the phrases “comprise,” “contain,” and “encompass” are interpreted more broadly than exclusively.

[0172] Unless otherwise defined, all technical and scientific terms and acronyms used herein have the same meaning as commonly understood by those skilled in the art of the present invention. Any methods and materials similar or equivalent to those described herein may be used in the practice of the present invention, but preferred methods and materials are described herein.

[0173] The present invention is further illustrated by the following drawings and embodiments, which are intended to describe but not limit the invention, and from therein further features, embodiments, and advantages can be inferred. Thus, any specific modifications described herein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it will therefore be understood that such equivalent embodiments are included herein. [Examples]

[0174] Part 1 - pH-Buffer-Salt Screening Test To test the effects of pH, buffer type, and ionic strength on formulations containing high concentrations of Fc polymer molecules (CSL730), a CSF730 formulation with a protein concentration of 100 mg / mL was prepared. Then, protein stability was investigated.

[0175] CSL730 is a trivalent Fc polymer consisting of two long chains from SEQ ID NO: 24 and two short chains from SEQ ID NO: 23, manufactured as described in WO2015 / 168643A2. This yields a trivalent Fc polymer as shown in Figure 12.

[0176] material and method Materials: Table 1 lists the materials used in the examples, their catalog numbers, and suppliers.

[0177] [Table 1]

[0178] Preparation of formulation: The formulation was prepared using CSL730 (recombinant Fc macromer bulk purified protein (concentration: 146 mg / ml)) as the starting material. The bulk purified protein was dialyzed against each of the 12 buffers listed below. The pH of each buffer was measured after preparation. A 30% excess (mg protein) was applied to compensate for losses. The formulation was dialyzed against approximately 1 L of buffer for approximately 5 hours, followed by replacement with fresh buffer (approximately 1 L), and dialyzed overnight. Dialysis was performed in a 15 ml 10K MWCO cassette at 2–8°C. The following buffers were prepared for dialyzing: 1.20 mM glacial acetic acid, 50 mM sucrose, pH 4.5 2.20 mM glacial acetic acid, 50 mM sucrose, pH 5 3.20 mM glacial acetic acid, 50 mM sucrose, pH 5.5 4.20 mM histidine, 50 mM sucrose, pH 5 5.20 mM histidine, 50 mM sucrose, pH 5.5 6.20 mM histidine, 50 mM sucrose, pH 6 7.20 mM histidine, 50 mM sucrose, pH 6.5 8.20 mM histidine, 50 mM sucrose, pH 7 9.20 mM histidine, 50 mM sucrose, 100 mM NaCl, pH 6 10.20 mM citric acid monohydrate, 50 mM sucrose, pH 5.5 11.20 mM citric acid monohydrate, 50 mM sucrose, pH 6 12.50 mM sucrose, pH 5.3 (no buffer solution)

[0179] After dialysis, the protein concentration of each sample was measured. If a sample was found to be too diluted, it was concentrated by centrifugation using Amicon tubes (30 kDa fraction) to the target concentration of 100 ± 10 mg / mL. The protein concentration was then confirmed.

[0180] The filling and final processes were performed under a laminar flow hood. Each formulation was filtered through a 0.22 μm filter, and 1 ml of the formulation was filled into a labeled 2 ml glass vial. The vial was then stoppered and crimped.

[0181] A summary of the formulations prepared for this study is shown in Table 2. The target buffer concentration for all formulations was 20 mM. Acetic acid, histidine, and citrate buffers were used at different pH levels to achieve good buffering performance within the pH range of 4.5–7. No surfactants were added to the formulations in this study. 50 mM sucrose was added as a stabilizer to all formulations to provide a minimum level of stability. Note that sucrose does not affect ionic strength. One formulation without a buffer was prepared because it is known that high concentrations of protein self-buffer. The pH of the buffer-free solution would depend on the protein itself.

[0182] [Table 2]

[0183] After filling, the filled formulations were placed in a stabilization chamber at 2–8°C, 25°C, or 35°C. Samples were analyzed immediately after the filling / final step (for "t0" / t=0 analysis) and at predetermined subsequent time points up to 4 weeks later. To compare the degradation dynamics between different formulations, the rate constants (with standard error) of degradation via different pathways were measured using multiple linear regression over a minimum of three time points (t=0, t=2 weeks, and t=4 weeks). Multiple linear regression was performed using a general linear model (GLM), and the assay data were plotted and fitted to the model (Equation 1). %P=Po+kt[Formula 1]

[0184] Methods used to analyze the stability of the formulation: Stability was demonstrated by analyzing formulations at different time points, primarily using size exclusion chromatography (SEC), cation exchange chromatography (CEX), and capillary gel electrophoresis (CGE) (Caliper). Furthermore, the pH and appearance (color, turbidity, and visible particles) of the formulations were monitored at different time points.

[0185] Table 3 summarizes the methods used in this study, the purpose of each method, and the analyses performed at each stage of the analysis.

[0186] [Table 3]

[0187] The analysis method is described below:

[0188] Visual inspection: Visual inspection was performed in an inspection station equipped with a white and black background and fluorescent lighting. The formulation in the vial was gently agitated to avoid creating bubbles, and then inspected for color, clarity, and the presence of visible particles. The inspection was performed by two independent inspectors.

[0189] pH measurement: pH was measured using a Mettler Toledo SevenExcellence pH meter equipped with an InLab® Ultra Micro ISM electrode.

[0190] Ultraviolet spectroscopy: Protein concentration was measured using an IMPLEN P360 Nanophotometer with A280 / UV measurement of the undiluted formulation. Three measurements were performed per formulation, and the average value was calculated.

[0191] Size exclusion chromatography (SEC)-high-performance liquid chromatography (HPLC): Protein aggregation profiles of formulations were determined using SEC-HPLC. Samples were analyzed using a Dionex system (Ultimate3000) equipped with an Acquity BEH200 column (Waters, 4.6 × 150 mm). Samples were diluted to 10 g / L in appropriate buffer, injected at a rate of 0.3 mL / min under isocratec conditions, and separation was performed. The mobile phase consisted of BET buffer (pH 6.5) with a runtime of 15 minutes. Intact proteins were detected at 280 nm with a retention time of approximately 3.5 minutes. Monomeric species, high molecular weight species (HMWS, aggregates), and low molecular weight species (LMWS, fragments) were reported as relative area %. Runs were evaluated using internal and external references.

[0192] Cation exchange chromatography (CEX): CEX-HPLC was used to determine the proportions of acidic, major, and basic protein species. Samples were analyzed using a Dionex system (Ultimate3000) equipped with a Propack WCX-10 column (Thermo Fisher, 4 × 250 mm). Samples were diluted to 10 g / L in appropriate buffer and separated using a gradient method at 0.7 mL / min with a 3.0 μL injection volume. Briefly, two aqueous buffers (imidazole, piperazine, and TRIS-based) at pH 5 and pH 10 were changed over 55 minutes. Species were detected at 280 nm, identified against a reference standard, and reported as a relative area percentage of the integrated area.

[0193] Capillary gel electrophoresis (CGE) "Caliper" method: Protein "binding patterns" were obtained by capillary gel electrophoresis. Analysis was performed using a microfluidic LabChip GXII system (Perkin Elmer Australia Pty Ltd). Protein electrophoresis on a microfluidic chip was achieved by integrating the main features of one-dimensional SDS-PAGE: these include separation, staining, destaining, and detection. Denatured proteins were loaded directly onto the chip from a microtiter plate through a capillary sipper. The samples were then electrokinetically loaded and injected into a 14 mm long separation channel containing a low-viscosity matrix of entangled polymer solution. The entire sample preparation procedure was performed according to the manufacturer's protocol. For non-reducing samples, the protein solution was diluted to 1 g / L with buffer and Milliq water. Reducing samples were diluted with 1 M DTT. Denaturation occurred at 40°C for 20 minutes in the non-reducing sample and at 80°C for 15 minutes in the reducing sample. For the non-reducing sample, the results were reported as the relative area percentage of intact LMWS and HMWS. For the reducing sample, the long-chain and short-chain fractions were examined.

[0194] Example 1 Visual inspection at t=0 The appearance of all formulations was evaluated immediately before filling. Therefore, all filled vials of all formulations were evaluated by their appearance after filling / final processing.

[0195] Due to the high protein concentration, all formulations exhibited a slightly milky, brownish-yellow hue. The milkiness was observed to increase with pH. CSL730 in formulations at pH 6.5 and pH 7 precipitated more strongly at pH 7. It is well known that higher pH levels can be used to precipitate Fc fragments. The precipitated fragments were subsequently recovered by solubilization in a low pH buffer. This was confirmed by the precipitated portion of recombinant Fc polymers (data not shown). However, the effect of this high pH exposure on storage stability is not well understood.

[0196] All formulations (including precipitated formulations) were filtered before filling vials and re-tested after stoppering and crimping. All formulations were brownish-yellow, slightly opaque, and free of visible particles (including pH 6.5 and pH 7.0 formulations). Gelation was not observed in any of the prepared formulations.

[0197] Overall, the results showed that formulations with a pH of ≥ 6.5 resulted in protein precipitation. Formulations with a lower pH value, down to 4.5, were more preferable because the lower pH did not result in protein precipitation.

[0198] Example 2 pH and protein concentration measurement at t=0 pH and protein concentration measurements were performed at t=0 for all formulations 1-12 (see Table 2). The difference from the target value was also evaluated. The results are summarized in Table 4.

[0199] pH measurements showed that, with the exception of the acetate formulation with a target pH of 4.5 and the histidine formulation with a target pH of 5.0, the measured pH values ​​were within ±0.2 pH units of the target pH for most formulations, with the exception of the acetate formulation with a target pH of 4.5 and the histidine formulation with a target pH of 5.0, whose pH values ​​were within 0.4 and 0.3 units of the target pH, respectively. Overall, based on the measured pH values, the pH screening in this study covers a pH range of 4.88 to 6.92.

[0200] Regarding the measured protein concentrations, most formulations were within the target concentration of 100 + / - 10 mg / mL. The exceptions were the acetic acid formulations with target pH 4.5 and 5.0. These samples were overdiluted due to error, which is considered a test deviation. On the other hand, the histidine formulations with pH 6.5 and 7.0 had low concentrations as a result of precipitation.

[0201] The results indicate that the pH values ​​of the formulations were as expected in most cases. Except for the acetate formulation, the significant decrease in protein concentration at pH ≥ 6.5 is associated with significant protein precipitation under these conditions.

[0202] [Table 4]

[0203] Example 3 High molecular weight (HMW) profile at t=0 obtained by size exclusion chromatography All formulations 1-12 (see Table 2) were analyzed by size exclusion chromatography to quantify the high molecular weight (soluble aggregate) species at t=0, as described above.

[0204] The soluble aggregate profiles of all formulations showed relatively low %HMW (overall <1.5%) for all formulations, with a general trend indicating that increasing pH is associated with a higher % of HMW molecules (up to the point of precipitation). Interestingly, the initial %HMW of the filtered solution of the formulations after precipitation at pH ≥ 6.5 was also less than 1.5%. This suggests that pH ≥ 6.5 is not suitable for formulation Fc polymer molecules at high protein concentrations, but may be suitable for formulation recombinant Fc polymer molecules at low protein concentrations.

[0205] Example 4 Low molecular weight (LMW) profile at t=0 by capillary gel electrophoresis All formulations 1-12 (see Table 2) were analyzed by non-reducing capillary gel electrophoresis using a caliper (NR Caliper) to quantify low molecular weight species (fragments) at t=0, as described above.

[0206] All formulations except formulation 8 (which precipitated at pH ~7) showed relatively low %LMW (1.5~2.0%). Overall, the data did not show a proportional relationship between pH and %LMW. However, the increase in %LMW at pH 6.9 suggests that there may be a threshold pH at which a more measurable increase in %LMW occurs.

[0207] Example 5 Acidic and basic species profiles at t=0 obtained by cation exchange chromatography (CEX) As described above, all formulations were analyzed by cation exchange chromatography (CEX) to quantify the acidic and basic species at t=0.

[0208] With the exception of formulations 7 and 8, all formulations showed comparable levels of % acidic species. The percentage of acidic species was lowest in formulation 7 (52%) and lowest in formulation 8 (45%).

[0209] The data did not show a relationship between pH and the initial % acidic species. The decrease in the initial % acidic species in histidine buffer at pH ≥ 6.5 suggests that there may be a threshold pH beyond which a significant decrease in acidic species is observed.

[0210] With the exception of formulations 7 and 8, all formulations showed comparable levels of % basic species. Compared to all formulations, formulations 7 and 8 had a lower percentage of basic species (4%).

[0211] The data did not show a relationship between pH and initial % basic species. The decrease in initial % basic species in histidine buffer at pH ≥ 6.5 suggests that there may be a threshold pH beyond which a decrease in basic species is observed.

[0212] However, the results for formulations 7 and 8 are affected by protein precipitation of both acidic and basic species, as described above.

[0213] Summary of Examples 1-5 Based on analyses performed during processing and immediately after the filling / final step (T=0), it can be concluded that formulations with a pH of ≥ 6.5 are not suitable for formulations of Fc polymer molecules at high concentrations (≥ 100 mg / mL).

[0214] Nevertheless, all formulations were made stable enough to cover the degradation kinetics across the pH range of all formulations.

[0215] Example 6 Visual inspection over time Formulations 1-12 (see Table 2) were visually inspected at 2 and 4 weeks. The results are summarized in Table 5.

[0216] As described above, the two inspectors (Insp1 and Insp2) examined the appearance at each time point and temperature. In most cases, there was general alignment between the inspectors.

[0217] At all storage temperatures, no changes were observed in the color and clarity of most formulations. All formulations maintained a slightly brownish-yellow color, while their clarity remained slightly milky white. Formulations at higher storage temperatures began to show visible particles, with the most pronounced particle formation occurring at pH ≥ 6.5. Interestingly, formulations containing salt did not show visible particles compared to the same formulation at pH 6.

[0218] The results indicate that particle formation at each time point of visual inspection increased at pH ≥ 6.5 compared to samples with a lower pH.

[0219] [Table 5]

[0220] Example 7 pH and protein concentration over time pH and protein concentration were monitored for formulations 1-12 (see Table 2) at 2-8°C, 25°C, and 35°C for up to 4 weeks.

[0221] Overall, within experimental margins of error, the results showed minimal shifts in pH and protein concentration for all formulations at 2–8°C, 25°C, and 35°C during storage for up to 4 weeks.

[0222] Example 8 Relationship between high molecular weight (HMW) formation over time and pH / buffer / salt (size exclusion chromatography) %HMW species were monitored for formulations 1–12 (see Table 2) at 2–8°C, 25°C, and 35°C for up to 4 weeks. A representative plot of the change in %HMW over time for formulations stored at 35°C is shown in Figure 1. Figure 1 shows an initial rapid increase in %HMW species followed by a slow increase over time. The same trend was observed for formulations stored at 25°C.

[0223] Table 6 shows the percentage change in agglutination species (%HMW) after 4 weeks of storage at 2–8°C. The agglutination rate constant, along with the corresponding standard error (SE), was determined for all formulations at 25°C and 35°C (from 3 time points) using multiple linear regression with Graphpad Prism software. The results are summarized in Table 6.

[0224] The results in Table 6 show the rate of aggregation, which increased with temperature and pH. Faster aggregation rates occurred at pH ≥ 6.0. The trend was more pronounced at 35°C. The formulation in histidine buffer at pH 6.9 (Formulation 8) appeared to be the most stable. However, compared to the higher protein concentrations of all other formulations, this is an artifact of the very low protein concentration (18 mg / mL) in this formulation after protein precipitation (see Table 4).

[0225] The type of buffer had little effect on the aggregation rate. At equivalent pH, different buffers performed similarly. The self-buffered formulation (formulation 12) was slightly less stable than the formulation with buffer at equivalent pH (formulation 5). The addition of salt (which results in higher ionic strength) did not have a significant effect on improving the aggregation behavior.

[0226] The aggregation trend is summarized in Figure 2, which shows pooled data (aggregation rates at 25°C and 35°C) as a function of measured pH (excluding pH 7 formulations where aggregation behavior is not typical). Figure 2 shows the overall nonlinear increase in the aggregation rate with increasing measured pH.

[0227] [Table 6]

[0228] Example 9 The relationship between the formation of fragments (LMW) over time and pH / buffer / salt (non-reducing caliper) Fragmentation of formulations 1-12 (see Table 2) was monitored for up to 4 weeks at 2-8°C, 25°C, and 35°C. A representative plot of the change in % fragmentation over time for formulations stored at 35°C is shown in Figure 3. Figure 3 shows an initial rapid increase in %LMW species (fragments), followed by a slow increase in species over time. The same trend was observed for formulations stored at 25°C.

[0229] Table 7 shows the percentage change in fragmentation after 4 weeks of storage at 2–8°C. The fragmentation rate constant, along with the corresponding standard error (SE), was determined for formulations at 25°C and 35°C (from 3 time points) using multiple linear regression with Graphpad Prism software. Multiple linear regression was performed using a general linear model (GLM), and the assay data were plotted and fitted to the model (Equation 1). The results are summarized in Table 7.

[0230] The data revealed that fragmentation occurring at 2–8°C was typically low. However, the rate of fragmentation increased with storage temperature and pH (pH ≥ 6.0). A more significant change in %LMW was observed at 2–8°C with formulations at ~pH 7. At 25°C and 35°C, the rate of fragmentation was also highest with formulation 8 at ~pH 7 (despite the lowest protein concentration of 18 mg / mL), followed by pH 6.5. At the same pH, the type of buffer had only a small effect on the aggregation rate. No clear advantage was found with the addition of NaCl (high ionic strength).

[0231] The fragmentation trend is summarized in Figure 4, which shows pooled data as a function of measured pH (fragmentation rate at 35°C with standard error). As observed in aggregation, Figure 4 shows that, overall, the rate of fragmentation increased with increasing measured pH (≥pH 6.0).

[0232] [Table 7]

[0233] Example 10 Relationship between the formation of acidic species over time and pH / buffer / salt (cation exchange chromatography) The formation of acidic species was monitored for up to 4 weeks in formulations 1–12 (see Table 2) at 2–8°C, 25°C, and 35°C. A representative plot of the change in % acidic species over time for formulations stored at 35°C is shown in Figure 5. Figure 5 shows a more linear increase in % acidic species over time compared to the plots of % HMW (Figure 1) and % LMW (Figure 3) (except for the histidine formulation at ~pH7). The same trend was observed for formulations stored at 25°C.

[0234] Table 8 shows the percentage change in acidic species after 4 weeks of storage at 2–8°C. The rate constant of acidic species formation, along with the corresponding standard error (SE), was determined for formulations at 25°C and 35°C (from 3 time points) using multiple linear regression with Graphpad Prism software. Multiple linear regression was performed using a general linear model (GLM), and assay data were plotted and fitted to the model (Equation 1). The results are also summarized in Table 8.

[0235] The data revealed that fragmentation occurring at 2–8°C was generally low. However, the rate of acidic species formation increased with storage temperature and pH (pH ≥ 6.0). In formulations at ~pH 7, the highest levels of acidic species were formed after 4 weeks at 2–8°C. At 25°C and 35°C, the rate of acidic species formation was also highest in formulation 8 at ~pH 7 (despite the lowest protein concentration of 18 mg / mL), followed by pH 6.5.

[0236] At the same pH, the type of buffer was also found to play a role in the rate of acidic species formation, with formulations in histidine buffer showing greater storage stability compared to formulations in acetic acid or citrate buffer at the same pH.

[0237] The addition of NaCl (high ionic strength) resulted in greater storage stability in terms of the rate of acidic species formation compared to formulations of the same pH without salt.

[0238] The trend of acidic species formation with respect to pH is summarized in Figure 6, which shows pooled data as a function of measured pH (rate of acidic species formation at 25°C and 35°C). As observed by aggregation and fragmentation, Figure 6 shows that, overall, the rate of acidic species formation increases as the measured pH increases (>pH 6.0).

[0239] [Table 8]

[0240] Conclusions of Examples 1-10 The results indicated that formulations of high-concentration (100 mg / mL) Fc polymer molecules (CSL730) require specific conditions to prevent protein precipitation. pH ≥ 6.5 was shown to be unsuitable for high-concentration (100 mg / mL) Fc polymer molecule (CSL730) formulations due to precipitation. Formulations of high-protein-concentration (100 mg / mL) Fc polymer molecule (CSL730) formulations were more stable against aggregation, fragmentation, and acidic species formation at pH levels below 6. Furthermore, formulations of high-protein-concentration (100 mg / mL) Fc polymer molecule (CSL730) formulations were more stable against acidic species formation with high salt concentrations and histidine buffer.

[0241] Part 2 - Stabilizer Screening Test Excipient screening tests were conducted to investigate the effects of different stabilizers (sugars / polyols and amino acids), stabilizer levels, surfactant levels, antioxidant types, and antioxidant levels in liquid formulations containing Fc polymer molecules (CSL730) at protein concentrations of 10–100 mg / ml. Parallel short-term (3-month) tests were performed to examine storage stability as a function of protein concentration. Overall, CSL730 formulations with protein concentrations ranging from 10–100 mg / mL were prepared, and their protein stability was investigated.

[0242] material and method Materials: The materials used for the examples were the same as those used in Part 1; additional materials used in this part, their catalog numbers, and suppliers are listed in Table 9.

[0243] [Table 9]

[0244] Preparation of formulation: The formulation was prepared from 120 mg / mL of CSL730 recombinant Fc multimer bulk purified protein formulated in 20 mM histidine, 40 mM NaCl, and 200 mM proline at pH 6.0.

[0245] Histidine was the selected buffer in all formulations at a target buffer concentration of 20 mM. pH was fixed in all formulations, except for one formulation which was fixed at 5.25 ± 0.1. Polysorbate 80 (PS80) was the selected surfactant in all formulations. PS80 concentration was fixed in all formulations, except for one formulation which was fixed at 0.02% w / v. Different stabilizers (proline, arginine, sucrose, trehalose, and sorbitol) were used across the target concentration range of 200–300 mM. Four formulations contained different levels (1–20 mM) of antioxidants (either methionine or reduced glutathione GSH). The tests (and therefore the differences in formulations) were designed primarily to modify the one-of-a-kind method (OFAT). A summary of the formulations prepared for this test is shown in Table 10.

[0246] The following buffer solutions were prepared for dialysis: 1.20 mM histidine, 250 mM sucrose, pH 5.25 2.20 mM histidine, 250 mM trehalose, pH 5.25 3.20 mM histidine, 250 mM sorbitol, pH 5.25 4.20 mM histidine, 250 mM arginine, pH 5.25 5.20 mM histidine, 167 mM arginine, 83 mM sucrose, pH 5.25 6.20 mM histidine, 225 mM sucrose, pH 5.25

[0247] To prepare the formulations (2 - 6, 8 - 16, 18), approximately 30 - 50 mL portions of the bulk purified protein were dialyzed against each of the six buffers described above. The pH of each buffer was measured after preparation. More than 30% (mg protein) was applied to compensate for losses. The formulations were dialyzed against approximately 1 L of buffer for about 5 hours, followed by replacement with fresh buffer (about 1 L) and dialysis overnight. Dialysis was performed at 2 - 8 °C in 30 or 70 ml 10K MWCO cassettes.

[0248] After dialysis, the protein concentration of each dialysate was measured. If samples were found to be too diluted, they were concentrated by centrifugation using Amicon tubes aiming for 120 ± 5 mg / mL. The protein concentration was then confirmed.

[0249] The following stock solutions were prepared and used as spike solutions to assist in the preparation of the final formulations: 1. 20 mM histidine, 5.75% w / v (500 mM) proline, 0.02% w / v PS80, pH 5.25 2. 20 mM histidine, 17.04% w / v sucrose, 0.02% w / v PS80, pH 5.25 3. 10% w / v PS80 4. 20 mM histidine, 200 mM proline, 40 mM sodium chloride, 0.02% w / v PS80, pH 5.8 (labeled as "CSL730 diluent")

[0250] The active pharmaceutical ingredient (DS) consisted of 118 mg / mL recombinant Fc macromer CSL730, 20 mM histidine, 40 mM NaCl, 200 mM proline, and 0.02% w / v PS80 at pH 6. DS was diluted by adding an appropriate volume of stock solution 1 or stock solution 2 to a protein concentration of 100 ± 5 mg / mL, and then the pH was adjusted to 5.25 ± 0.1 using 0.1 M HCl to prepare formulations 1 and 7, respectively. Formulation 17 was prepared by direct dilution of DS to 100 ± 5 mg / mL using stock solution 4 while maintaining the pH at 5.8 ± 0.1.

[0251] After the concentration process and confirmation of protein concentration (A280 measurement), formulations 2-6, 8-16, and 18 were appropriately diluted with buffers 1-6 if necessary, and the final pH was adjusted to 5.25 ± 0.1 using 0.1 M HCl. Protein concentration was confirmed after the pH adjustment process.

[0252] Filling and final processing were performed under a laminar flow hood. Each formulation and its corresponding placebo were filtered through a 0.22 μm PES sterile filter, and 0.8 ml of the formulation was filled into a labeled 2 ml glass vial. The vial was then stoppered and crimped. In total, 17 liquid formulations and 13 placebos were prepared.

[0253] After filling / final processing, vials of the filled liquid formulation were placed in a stabilization chamber at 2–8°C, 25°C, or 40°C. For "T0" / T=0 analysis, samples were analyzed immediately after filling / final processing, and then at predetermined subsequent time points up to 6 months. To compare the degradation kinetics between different formulations, the rate constants (with standard error) of degradation via different pathways were measured using multiple linear regression with JMP and Excel software. Multiple linear regression was calculated using a general linear model (GLM), and assay data were plotted and fitted to the model (Equation 1). %P=Po+kt[Formula 1]

[0254] [Table 10]

[0255] Methods used to analyze the stability of the formulation: Stability was demonstrated by analyzing the formulations for oxidation at different time points, primarily using size exclusion chromatography (SEC), cation exchange chromatography (CEX), capillary gel electrophoresis (CGE) (Caliper), and reverse-phase HPLC (RP-HPLC). Furthermore, the pH, appearance (color, turbidity, and visible particles), and invisible particles (SVP) of the formulations were monitored at different time points.

[0256] The methods used in this study and the objectives of each method are summarized in Table 11. The storage conditions and analysis time points are summarized in Table 12.

[0257] [Table 11]

[0258] [Table 12]

[0259] The analysis method is described below: Visual inspection, pH measurement, ultraviolet spectroscopy, size exclusion chromatography, cation exchange chromatography, and capillary gel electrophoresis were performed as described in Part 1 above.

[0260] Osmotic pressure: The osmotic pressure of the formulation was measured at the start time (T0) by freezing point depression using a Gonotec Osmomat 3000 Osmometer. The sample volume was 300 μL. Measurements were performed three times per formulation, and the average value was calculated.

[0261] Testing of particle counts invisible to the naked eye: Limited analysis of particles invisible to the naked eye was performed on the target formulation at 2-8°C and 25°C, only after a 3-month storage period, using a FlowCam Biologics instrument (Dynamic / Flow Imaging Particle Analysis - DIPA technology). A minimum sample volume of 0.5 mL was used. Measurements were performed three times per formulation, and the average value was calculated.

[0262] Analysis of oxidation by reversed-phase high-performance liquid chromatography (RP-HPLC): Using the RP-HPLC method, the total amount of oxidized species and the relative amounts of oxidation of both short-chain and long-chain molecules were determined as a percentage of the total area. Sample preparation involved diluting the sample to 10 g / L in appropriate buffer, followed by the use of guanidine as a denaturing agent and DL-dithiothreitol (DTT) for the reduction of disulfide bonds. This step produced two identical long-chain molecules and two identical short-chain molecules. To prevent oxidation of the fragments, a subsequent dilution step with a solution containing a high concentration of L-methionine was performed, followed by sample analysis at a sample concentration of 2 μg / μl. Samples were analyzed using a Thermo Ultimate 3000 (or equivalent) equipped with an AdvanceBio RP-mAb Diphenyl 3.5 μm, 2.1 × 50 mm column. Separation was performed by gradient spectroscopy at 0.35 ml / min using a 3 μL injection volume. The column temperature was set to 65°C. In short, two buffer solutions (0.1% trifluoroacetic acid in water and 0.08% TFA in acetonitrile) were exchanged over a 20-minute period. Species were detected at 280 nm, identified against a reference standard, and reported as a relative area percentage to the integrated area.

[0263] Analysis of Polysorbate 80 (PS80): The amount of PS80 at the starting point (T0) of different formulations was quantified using RP-HPLC. Briefly, PS80 standards and samples were treated with ethanol and then with 0.1 M KOH at 40 °C, and then sample analysis of oleic acid resulting from hydrolysis was performed by reverse-phase HPLC method. Samples were analyzed using a Dionex (Ultimate 3000) system (or equivalent) equipped with a Nova-Pak® C18 3.9×150 mm, 4 μm reverse-phase column (Waters). The injection volume was 15 μL, and separation was performed using an isocratic method at 2.0 ml / min. The mobile phase was 80% acetonitrile containing 20% potassium dihydrogen phosphate buffer at pH 2.8. The column temperature was set at 40 °C. The species was detected at 250 nm and quantified using a standard calibration curve prepared with a PS80 standard solution. The data was reported as %(w / v) of PS80.

[0264] Example 1 Visual inspection at T0 and over time All filled vials were examined by two inspectors (Insp1 and Insp2 above), and by appearance after filling / final process, at each time point and temperature (100% visual inspection). No significant differences were found in the visual descriptions among the inspectors.

[0265] Except for the 10 mg / ml formulation, all other formulations showed slightly brownish yellow (BY) with a slight opalescence (70 mg / mL formulation) to BY (100 mg / mL formulation) due to the high protein concentration. Visible particles of 0 to 2 were randomly observed in some formulations and the placebo. Findings on the protein revealed that the visible particles were natural and exogenous (probably introduced during filling / final process). The formulations with high protein concentration did not gel.

[0266] Formulations 1-18 (see Table 10) were visually inspected at different temperatures for up to 6 months. The color of most formulations remained unchanged from T0 at all storage temperatures (colorless for the 10 mg / ml formulation, slightly BY for the 70 mg / ml formulation, and BY for the 100 mg / ml formulation). Formulations containing reduced GSH were slightly more BY in color compared to formulations of the same protein concentration. Transparency remained unchanged (clear for the 10 mg / ml formulation, slightly milky for all others). Particle count remained unchanged from T0 (0-2) regardless of temperature and time (0-2). The formulations did not gel during storage at any temperature.

[0267] Over time, the GSH-containing placebo showed a slight discoloration to BY or BY color. The color was intensified with increasing storage time, storage temperature, and GSH concentration. Placebos of the other formulations remained clear and colorless regardless of temperature and time. Overall, the particle count remained unchanged from T0.

[0268] Example 2 Measurement of osmotic pressure, pH, protein concentration, and polysorbate 80 at T0 and over time. Osmotic pressure, pH, protein concentration, and polysorbate 80 (PS80) concentration were measured for all formulations 1-18 (see Table 10) at time of baseline (T0). The results are summarized in Table 13.

[0269] The results in Table 13 showed that the protein concentration was within 100 ± 5 mg / mL for the high-concentration formulations. pH measurements showed that the measured pH values ​​were within ±0.1 pH units of the target pH value for all formulations. Osmotic pressure measurements were generally within 300-390 mOsm / kg, except for arginine-containing formulations (formulations 5 and 6) where the osmotic pressure value exceeded 400 mOsm / kg. The PS80 concentration for all formulations was within the maximum value of ±0.003% w / v of the PS80 target concentration.

[0270] pH and protein concentration were monitored for formulations 1-18 at 2-8°C, 25°C, and 40°C for up to 6 months (see Table 10).

[0271] Table 14 summarizes the results of pH measurements at different storage temperatures and times. Within experimental error, the results showed minimal pH shift (maximum ±0.1 units) at all temperatures for all formulations within the measured time frame.

[0272] Table 15 summarizes the results of protein concentration measurements at different storage temperatures and times. Within experimental error, the results showed small shifts in protein concentration over time. Three formulations showed relatively large changes in protein concentration over time (F3 at 40°C and F15 at 25°C and 40°C), but no protein precipitation was observed in any of these formulations. The mean variation in protein concentration over the measurement period was 1.0 mg / mL at 2–8°C, 1.7 mg / mL at 25°C, and 1.6 mg / mL at 40°C. This is considered to be a result of the relatively high assay variability of the high-throughput technology used.

[0273] [Table 13]

[0274] [Table 14]

[0275] [Table 15]

[0276] Example 3 Limited analysis of particles invisible to the naked eye at 6 months For most formulations tested after 3 months of storage at both 5°C and 25°C across all tested particle size ranges, we measured the number of particles invisible to the naked eye (see Figure 7).

[0277] High particle counts, invisible to the naked eye, were observed at F1 (both 5°C and 25°C). This may be related to the method of producing the formulation. The high levels of particles are related to pH adjustment and spiking, not dialysis.

[0278] Example 4 Testing of aggregation behavior at T0 and over time by size exclusion chromatography (high molecular weight species) All formulations 1-18 (see Table 10) were analyzed by size exclusion chromatography to quantify high molecular weight (soluble aggregate) species at T0, as described above.

[0279] The baseline (T0)%HMW for all formulations is shown in Table 17. The aggregation rate constants, along with the corresponding standard error (SE), were determined for all formulations at 2–8°C, 25°C, and 40°C by multiple linear regression, as described above. The results are also summarized in Table 16.

[0280] The initial (T0) soluble aggregate profiles of all formulations showed variation in %HMW. This is attributed to differences in the preparation methods used for different formulations. For example, some dialysates had to be concentrated after dialysate, while others were not. Overall, relatively low %HMW was measured for all formulations at T0 (overall <1.5%). Interestingly, a systematic increase in aggregate (%HMW) levels was observed in formulations with increased protein concentration (F2, F10, F11). Note that these formulations were prepared by simple dilution using the same buffer from the same stock dialysate solution.

[0281] Analysis of all formulations was interrupted after 3 months of storage at 40°C because aggregation in all formulations reached a level sufficient to distinguish between formulation performance in terms of aggregation stability (from best to worst performance). Analysis of F10, F11, F14, and F15 was also interrupted after 3 months at 2–8°C and 25°C. Aggregation data recovered up to 3 months at 2–8°C and 25°C for F10 and F11 were sufficient to determine the effect of protein concentration on physical stability (due to aggregation). On the other hand, F14 and F15 showed weak stability sufficient to guarantee their exclusion by this method and other methods (discussed). In general, % major species (monomer) was observed to decrease over time with a simultaneous increase in aggregation (% HMW) and fragmentation (% LMW). However, fragmentation was not reported by this method but was reported by a more reliable method for LMW (CGE), as described later.

[0282] As expected, the results in Table 16 show an increased aggregation rate with increasing storage temperature. For every 10°C increase in temperature up to 40°C, the aggregation rate increased by an average of 1.75 times, and a more significant change was observed, suggesting the possibility of a different mechanism of molecular aggregation at higher storage temperatures compared to low-temperature storage conditions.

[0283] To evaluate the effect of different stabilizer types, the storage stability of F1-F7 was compared. The stabilizers used in the formulations were sugar / polyol, amino acids, or mixtures of amino acids and sugars. The total stabilizer level of these formulations was fixed at 250 mM. The results in Table 16 show that the Fc polymer molecule (CSL730) in sucrose and trehalose-based formulations (F2 and F3, respectively) had better storage stability compared to all other formulations at all temperatures, as evidenced by the slightly lower aggregation rates at 2-8°C and 25°C and the significantly lower aggregation rate at 40°C. The sorbitol-based formulation showed better stability than the amino acid-based formulation at 40°C (with or without sucrose containing amino acids), but showed comparable stability at lower temperatures up to 6 months. Similarly, the proline-based formulation showed better stability than the arginine-based formulation at 40°C (with or without sucrose), but showed comparable stability at lower temperatures up to 6 months.

[0284] To evaluate the effect of protein concentration at the fixation stabilizer level, the storage stability of F2, F10, and F11 was compared with sucrose as a sugar stabilizer. The protein concentration range was 10–100 mg / mL. As expected, the results in Table 16 show that the Fc polymer molecule (CSL730) exhibited excellent stability in F11 at a very low concentration (10 mg / mL), followed by 70 mg / mL (F10), and then 100 mg / mL (F2). Plots of the aggregation rate constant as a function of protein concentration for all three formulations at all temperatures demonstrated a nonlinear increase in aggregation rate with increasing protein concentration (results not shown). Note that at the fixation stabilizer level, the molar ratio of stabilizer to protein increases as protein concentration decreases (Table 10), and this may be a factor that contributes to a significant improvement in storage stability.

[0285] To evaluate the effect of stabilizer levels at fixed (high) protein concentrations (100 mg / mL), the storage stability of F2, F8, and F9 was compared with sucrose as a sugar stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 16 show that 225 mM (F8) sucrose was the least stable sucrose formulation compared to 250 mM (F2) and 300 mM (F9) formulations at all storage temperatures. F9 showed progressively better storage stability than F2 at all temperatures. Similarly, the Fc polymer molecule (CSL730) (F17) in 200 mM proline was significantly less stable than the formulation in 250 mM proline at all storage temperatures. Interestingly, the Fc polymer molecule (CSL730) was more stable than all sucrose formulations compared to the proline formulation at a storage temperature of 40°C.

[0286] To evaluate the effect of antioxidants (type and level) at the fixation stabilizer level, the storage stability of F2, F12, F13, F14, and F15 was compared. Methionine (10 and 20 mM) and reduced glutathione (GSH) (1 and 10 mM) were used as antioxidants. The results in Table 16 show that methionine as an antioxidant showed an increasing (small) improvement in storage stability (physical) of the Fc polymer molecule (CSL730) with increasing methionine levels at 40°C. However, there was no significant difference in the physical stability between formulations containing and not containing methionine. Formulations containing reduced GSH showed significantly greater instability at 40°C compared to formulations without antioxidants (F2) and formulations containing methionine as an antioxidant (F12, F13). A slight difference in storage stability was observed among the GSH-containing formulations compared to F2, F12, and F13 at 25°C, but the results were the same (lower stability with reduced GSH formulations). Nevertheless, the reduced GSH preparations (F14, F15) were excluded after 3 months of storage stability because stability data from other methods showed significant instability for F14 and F15, which guaranteed their exclusion.

[0287] To evaluate the effect of surfactant (PS80) levels at the fixation stabilizer level, the storage stability of F2 and F18 was compared. PS80 levels were evaluated at 0.02% w / V and 0.04% w / v. No significant difference in storage stability (aggregation) was observed between the two formulations at 2–8°C and 25°C up to 6 months. However, the Fc polymer molecule (CSL730) was more stable at the lower PS80 concentration (F2) at 40°C.

[0288] [Table 16]

[0289] Example 4 Testing of fragmentation behavior (low molecular weight species) at T0 and over time by non-reducing capillary gel electrophoresis (NR-cGE). All formulations (see Table 10) were analyzed by non-reducing cGE using a caliper (NR Caliper) to quantify low molecular weight (LMW) species (i.e., fragmentation) at T0, as described above. Placebo samples were also run, and no interference from the placebo component was observed in the cGE chromatogram due to protein peaks (data not shown).

[0290] The baseline (T0)% LMW total species was measured for all formulations. F15 had the highest T0 level of fragments, and this observation was also confirmed using SEC (data not shown). The baseline (total)% LMW species measured for all formulations except F15 were relatively low, ranging from 2.1% to 2.5%.

[0291] Except for F10 and F11, the fragmentation behavior of formulations was monitored over time at different temperatures using the NR-cGE "Caliper" method, as it is a relatively high-throughput method, in order to select the formulations. The fragmentation rate constants, along with the corresponding standard error (SE), were determined at different storage temperatures by multiple linear regression as described above. The results are also summarized in Table 17.

[0292] Analysis of all formulations was interrupted after 3 months of storage at 40°C because fragmentation of all formulations reached a level sufficient to distinguish between the performance of the formulations with respect to fragmentation stability (from best to worst performance). Analysis of F14 and F15 was also interrupted after 3 months at 2–8°C and 25°C, respectively, because they showed weak stability sufficient to guarantee their exclusion by this method and other methods (aggregation, oxidation). In general, as observed by SEC analysis, % major species were observed to decrease over time with a simultaneous increase in aggregation (% HMW) and fragmentation (% LMW). However, aggregation is not reported by this method because it was reported by SEC, which is a more reliable method for soluble aggregates.

[0293] Small variations in fragmentation (within assay error) occurred in all formulation tests over time at 2–8°C, as evidenced by very small changes in %LMW at 3 and 6 months.

[0294] As expected, the results in Table 17 show that the rate of fragmentation increases with storage temperature.

[0295] To evaluate the effect of different stabilizers, the storage stability of F1-F7 was compared. The results in Table 17 show that the Fc polymer molecule (CSL730) exhibited more significant fragmentation ability in the presence of arginine (with or without sucrose: F5 and F6) at both 25°C and 40°C. Fragmentation was comparable for all other formulations at 25°C. At 40°C, less fragmentation was observed in sucrose and sorbitol (F2 and F4, respectively) compared to proline and trehalose formulations (F1 and F3, respectively), as evidenced by the slower fragmentation rate at 40°C.

[0296] To evaluate the effect of stabilizer levels at a fixed protein concentration (100 mg / mL), the storage stability of F2, F8, and F9 was compared with sucrose as a sugar stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 17 did not show significant differences in fragmentation stability at 25°C as a function of sucrose levels. Surprisingly, the formulation with 250 mM sucrose (F2) showed better fragmentation stability at 40°C than the corresponding formulations with 225 mM and 300 mM sucrose. With proline as a stabilizer, the Fc polymer molecule (CSL730) (F17) in 200 mM proline showed slightly better fragmentation stability at 25°C and 40°C compared to the formulation with 250 mM proline.

[0297] To evaluate the effect of antioxidants (type and level) at the fixation stabilizer level, the storage stability of F2, F12, F13, F14, and F15 was compared. The results in Table 17 show that there was a small difference in the fragmentation storage stability of the Fc polymer molecule (CSL730) at 40°C in the presence versus absence of methionine as an antioxidant. Fragmentation stability was slightly better in the absence of methionine at 25°C for reasons that the inventors cannot explain. More significant instability with respect to fragmentation was observed only after 3 months of storage at both 25°C and 40°C with formulations containing reduced GSH. Therefore, the reduced GSH formulations (F14, F15) were excluded after 3 months of storage because the stability data showed sufficient instability for F14 and F15 to guarantee their exclusion by this method and other methods.

[0298] To evaluate the effect of surfactant (PS80) levels at the fixation stabilizer level, the storage stability of F2 (0.02% w / v PS80) and F18 (0.04% w / v PS80) was compared. No difference in storage stability (fragmentation) was observed between the two formulations at 25°C for up to 6 months. However, the Fc polymer molecule (CSL730) was more stable at the lower PS80 concentration (F2) at 40°C.

[0299] [Table 17]

[0300] Example 5 Testing of acidic and basic species at T0 and over time by cation exchange chromatography (CEX). All formulations (see Table 10) were analyzed by CEX for quantification of % acidic and basic species at T0, as described above. Placebo samples were also run by CEX, and no interference from the placebo component was observed in the CEX chromatogram due to protein peaks (data not shown).

[0301] Figure 8 compares the T0 (Time To) results for % acidic and basic species across all formulations. Most formulations showed comparable % acidic species at T0. F15 (high reduced GSH level) and F17 (low proline level) had approximately 2.5-3.5% lower initial % acidic species levels compared to the other formulations (see also Table 9). Similarly, most formulations showed comparable % basic species levels at T0 (approximately 4-5%). However, F15 had the highest initial % basic species level (6.1%) compared to the other formulations.

[0302] The % acidity of all formulations was monitored over time at different temperatures using CEX. The rate constant for acidity formation, along with the corresponding standard error (SE), was determined at different storage temperatures by multiple linear regression, as described above. The results are also summarized in Table 18.

[0303] Because the levels of acidic species in all formulations were sufficiently different and high to distinguish between the performance levels of the formulations with respect to the stability of acidic species (from best to worst performance), the analysis of all formulations was interrupted after 2 months of storage at 40°C. The analysis of F10, F11, F14, and F15 was also interrupted after 3 months at 2–8°C and 25°C. F14 and F15 showed weak stability sufficient to guarantee their exclusion by this method and other methods. In general, it was observed that the % major species decreased over time with a simultaneous increase in % basic species and a greater increase in % acidic species.

[0304] F15 showed a significant increase in % acidic species only after 3 months of storage at 2–8°C, followed by F17. Small variations in % acidic species (within assay error) were observed in other formulations at the same temperature. For formulations continued beyond 3 months at 2–8°C, small variations in % acidic species were observed within the same formulation. Among these formulations, F17 still showed high levels of % acidic species after 6 months of storage at 2–8°C (Table 18).

[0305] As expected, the results in Table 18 show the rate of acidic species formation increased with storage temperature.

[0306] To evaluate the effect of different stabilizers, the storage stability of F1-F7 was compared. The results in Table 18 show that the Fc polymer molecule (CSL730) exhibited surprisingly good stability in terms of the rate of acidic species formation in the presence of arginine (F5 and F6) at both 25°C and 40°C (different from the trends in fragmentation and aggregation stability).

[0307] To evaluate the effect of protein concentration at the fixation stabilizer level, the storage stability of F2, F10, and F11 was compared with sucrose as a sugar stabilizer. The results in Table 18 show that the Fc polymer molecule (CSL730) showed small differences in stability with respect to acid species formation among the three formulations with different protein concentrations at both 25°C and 40°C.

[0308] To evaluate the effect of stabilizer levels at a fixed protein concentration (100 mg / mL), the storage stability of F2, F8, and F9 was compared with sucrose as a sugar stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 18 show that the Fc polymer molecule (CSL730) showed small differences in stability with respect to acidic species formation among the three formulations with different sucrose levels at both 25°C and 40°C. Despite the high initial level of % acidic species in F17, the Fc polymer molecule (CSL730) (F17) in 200 mM proline was comparable in stability with respect to acidic species formation in 250 mM proline at both 25°C and 40°C. Overall, the stability of the Fc polymer molecule (CSL730) with respect to acidic species formation in sucrose and proline formulations was comparable at both 25°C and 40°C.

[0309] To evaluate the effect of antioxidants (type and level) at the fixation stabilizer level, the storage stability of F2, F12, F13, F14, and F15 was compared. The results in Table 18 show that there was a small difference in storage stability with respect to acidic species formation of the Fc polymer molecule (CSL730) in the presence versus absence of methionine as an antioxidant at both 25°C and 40°C. More instability in acidic species formation was observed in formulations containing reduced GSH, as well as in stability observations of aggregation and fragmentation. A systematic increase in the rate constant of acidic species formation up to 3 months at 25°C and up to 2 months at 40°C was accompanied by an increase in reduced GSH content. Therefore, the reduced GSH formulations (F14, F15) were excluded after 3 months of storage stability because the stability data showed sufficient instability for F14 and F15 to guarantee their exclusion by this method and other methods.

[0310] To evaluate the effect of surfactant (PS80) levels at the fixation stabilizer level, the storage stability of F2 (0.02% w / v PS80) and F18 (0.04% w / v PS80) were compared. Small differences were observed between the storage stability (acid species formation) of the two formulations at 25°C and 40°C.

[0311] [Table 18]

[0312] Example 6 Testing of oxidation behavior at T0 and over time by reversed-phase high-performance liquid chromatography (RP-HPLC). All formulations (see Table 10) were analyzed by RP-HPLC for the quantification of total % oxidation at T0 and % oxidation in long-chain (LC) and short-chain (SC) molecules, as described above. Figure 9 shows a representative chromatogram of the oxidation profile of the Fc polymer molecule (CSL730) in the sample formulations. Figure 9 shows the elution of SC followed by SC, then LC followed by LC. The chromatogram profile also includes SC and LC fragments, as well as SC and LC N tertiary pyruvate. Placebo samples were also run by RP-HPLC, and no interference from the placebo component was observed in the chromatogram due to protein peaks (data not shown). This was particularly important for the runs of formulations containing reduced GSH.

[0313] Figure 10 compares the results at baseline (T0) for the relative area (%) of %SC oxidized species, %LC oxidized species, and total oxidation for all formulations. The % oxidized species at T0 were comparable for most formulations. The baseline levels of the relative area (%) of %SC oxidized species, %LC oxidized species, and total oxidation were also comparable for most formulations. However, high levels of oxidation were observed in all measurements for formulations with reduced GSH (F14 and F15) (see also Table 19). As the reduced GSH level increased, the % oxidized species (SC and LC) and total % oxidation at T0 increased.

[0314] The relative area (%) of %SC oxidized species, %LC oxidized species, and total oxidation for all formulations was monitored over time at different temperatures using RP-HPLC. The rate constant of total oxidation, along with the corresponding standard error (SE), was determined at different storage temperatures by multiple linear regression, as described above. The results are also summarized in Table 19.

[0315] A small selection of formulations were analyzed at 2–8°C and 25°C for up to 6 months. Oxidation analysis of some formulations was interrupted after only 2 months at 25°C (based on their performance at 40°C). Analysis of all formulations was interrupted after 2 months of storage at 40°C because oxidation had reached sufficiently high levels to allow for the identification and exclusion of the formulations with the worst performance. In general, % non-oxidized SC and % non-oxidized LC were found to decrease over time with a simultaneous increase in % oxidized SC and % oxidized LC species. Overall, total % oxidation in the different formulations increased over time and at a rate that varied with temperature, as demonstrated in Figure 11.

[0316] Table 19 shows the formulations tested for oxidation after 6 months of storage at 2–8°C, and they showed a small increase in total % oxidation. However, the formulation containing arginine as the basic stabilizer (F5) showed the maximum oxidation after 6 months of storage, and F14 and F15 were not included in the 2–8°C range.

[0317] As expected, the results in Table 19 show that the rate of oxidation increased with storage temperature. In particular, the increase in the rate of oxidation at 25°C and 40°C was very slight in the presence of 10 mM reduced GSH (F15). This may indicate that at high levels of GSH in the tested system, oxidation becomes less temperature-dependent and more GSH-driven, thus leading to a change in oxidation kinetics.

[0318] To evaluate the effect of different stabilizers, the storage stability of F1-F7 was compared. The results in Table 19 show that the Fc polymer molecule (CSL730) was more stable against oxidation than amino acids at 25°C in the presence of polyols / sugars. However, the outcome at 40°C did not coincide with the outcome at 25°C. Therefore, no trend could be drawn from the results. Considering the assay variability at the measured oxidation levels, the differences between formulations are likely small.

[0319] To evaluate the effect of protein concentration at the fixation stabilizer level, the storage stability of F2, F10, and F11 was compared with sucrose as a sugar stabilizer. The results in Table 19 show that the Fc polymer molecule (CSL730) was more stable against oxidation, as indicated by increased protein concentration at both 25°C and 40°C. This appeared to be particularly true at 40°C when comparing the stability of the 10 mg / mL protein formulation (F11) to the 100 mg / mL and 70 mg / mL protein formulations (F2 and F10, respectively). F2 and F10 were 2.4 to 2.8 times more stable than F11 at 40°C.

[0320] To evaluate the effect of stabilizer levels at a fixed protein concentration (100 mg / mL), the storage stability of F2, F8, and F9 was compared with sucrose as a tower stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 19 show that the Fc polymer molecule (CSL730) exhibited slight differences in oxidation stability among the three formulations with different sucrose levels at both 25°C and 40°C. The same outcome applied in the presence of proline as a stabilizer at levels of 200 mM and 250 mM at both 25°C and 40°C.

[0321] To evaluate the effect of antioxidants (type and level) at the fixed-stabilizer level, the storage stability of F2, F12, F13, F14, and F15 was compared. The results in Table 19 show that increasing the level of methionine at 25°C resulted in a small but systematic improvement in oxidation stability compared to its absence (F12 and F13). A systematic but more significant improvement in oxidation stability was observed at 40°C with increasing levels of methionine. In contrast to formulations containing methionine (or even other formulations without antioxidants), formulations containing reduced GSH showed the weakest stability against oxidation (see also Figure 11). A nonlinear increase in the oxidation rate constant with increasing reduced GSH content was observed for up to two months of storage at both 25°C and 40°C. At both temperatures, just 1 mM GSH resulted in 4.7–9.3 times oxidation compared to its equivalent formulation with methionine at both 25°C and 40°C. Therefore (as with the other methods discussed earlier), reduced GSH preparations (F14, F15) were excluded based solely on their oxidative storage stability of 2 months.

[0322] To evaluate the effect of surfactant (PS80) levels at the fixation stabilizer level, the storage stability of F2 (0.02% w / v PS80) and F18 (0.04% w / v PS80) were compared. No measurable difference was observed between the storage stability (oxidation) of the two formulations at 40°C.

[0323] [Table 19]

[0324] Conclusion of Part Two A stable liquid DP formulation with a high concentration (≧100 mg / mL) of CSL730 is suitable. • Stable against aggregation, acid species formation, fragmentation, and oxidation. • In particular, factors that contribute to the stabilization of Fc polymer molecules at high protein concentrations. ·Low pH (5~5.8) • Use of disaccharides / polyols as stabilizers, and low levels of amino acids. The optimal molar ratio for protein stability is a protein-to-sucrose ratio of 1:420. Stability was also achieved with a low protein-to-sucrose ratio. The strength of the formulation increases when this ratio is exceeded. Antioxidants are required to prevent yellowing and / or side-chain oxidation reactions that may occur due to other excipients during storage, or to protect against certain manufacturing conditions (e.g., exposure to light sources, exposure to evaporated hydrogen peroxide residue from sterilization of isolator filling machines), or unusual handling conditions during clinical administration. • High PS80 levels are not required for storage stability, but are necessary to ensure stability during manufacturing and clinical handling / administration.

[0325] Part 3: Stabilizer screening test at protein concentrations of 120-160 mg / ml A follow-up study of the excipient screening test was conducted to investigate the effect of different stabilizers (based on Part II, the target stabilizers were sucrose, trehalose, and proline) on liquid formulations containing Fc polymer molecules (CSL730) at high protein concentrations in the presence of 20 mM histidine, 0.02% w / v polysorbate 80, and 10 mM methionine. Although the protein concentration varied, the molar ratio of protein to stabilizer was fixed at approximately 1:420. The pH of all formulations was fixed at 5.25 ± 0.1. Overall, CSL730 formulations with protein concentrations ranging from 120 to 160 mg / mL were prepared, and their protein stability was investigated. The formulations prepared for this study are summarized in Table 20.

[0326] [Table 20]

[0327] Methods used to analyze the stability of the formulation Stability was demonstrated by analyzing the formulations for oxidation at different time points, primarily using size exclusion chromatography (SEC), cation exchange chromatography (CEX), capillary gel electrophoresis (CGE) (Caliper), and reverse-phase HPLC (RP-HPLC). Furthermore, the pH, appearance (color, turbidity, and visible particles), and invisible particles (SVP) of the formulations were monitored at different time points.

[0328] The methods used in this study and the purpose of each method are summarized in Table 21. The storage conditions and analysis time points to date are summarized in Table 22.

[0329] [Table 21]

[0330] [Table 22]

[0331] The analysis method was carried out as described in Parts 1 and 2 above.

[0332] Example 1 Appearance at T0 and over time All filled vials were inspected by two inspectors (Insp1 and Insp2, as described above) and by appearance after filling / final process at each time point and temperature (100% visual inspection). In most cases, there was general alignment among the inspectors.

[0333] Due to the high protein concentration, all formulations exhibited a slightly milky, brownish-yellow (BY) color. In some formulations and the placebo, 0–1 visible particles were randomly observed. Further examination, based on the protein findings, revealed that the visible particles were naturally exogenous (likely introduced during the filling / final processing stage). Despite the high viscosity, no gelation of the formulations was observed.

[0334] Formulations 1-6 (see Table 20) were visually inspected at different temperatures for up to 12 months. The color of all formulations remained unchanged from T0 at all storage temperatures (BY). Transparency remained unchanged (slightly milky). Particle count remained unchanged from T0 regardless of temperature and time (0-1). The formulations did not gel during storage at any temperature.

[0335] Example 2 Measurement of osmotic pressure, pH, protein concentration, and polysorbate 80 at T0 and over time. Osmotic pressure, pH, protein concentration, and polysorbate 80 (PS80) concentration were measured for all formulations 1-6 (see Table 20) at time of baseline (T0). The results are summarized in Table 23.

[0336] The results in Table 23 show that protein concentrations were within 120 ± 5 mg / mL for F1, F3, and F5, and within 160 ± 5 mg / mL for F2, F4, and F6. pH measurements showed that the measured pH values ​​were within ±0.1 pH units compared to the target pH value for most formulations. A slightly higher pH deviation was observed in F6 (within 0.17 units). Osmotic pressure was typically within 382–414 mOsm / kg for the 120 mg / mL formulation, but was generally higher for the 160 mg / mL formulation (529–551 mOsm / kg). The PS80 concentration for all formulations was within the maximum value of ±0.003% w / v of the PS80 target concentration.

[0337] pH and protein concentration were monitored for formulations 1–6 at 2–8°C, 25°C, and 35°C for up to 12 months (see Table 20). Within experimental error, the results showed minimal pH shifts (maximum ±0.1 units) at all temperatures for all formulations within the measured time frame. Within experimental error, the results also showed small shifts in protein concentration over time.

[0338] [Table 23]

[0339] Example 3 Testing of aggregation behavior (high molecular weight species) at T0 and over time by size exclusion chromatography. All formulations 1-6 (see Table 20) were analyzed by size exclusion chromatography to quantify high molecular weight (soluble aggregate) species at T0, as described above.

[0340] The baseline (T0)%HMW for all formulations is shown in Table 24. The aggregation rate constants, along with the corresponding standard error (SE), were determined for all formulations at 2–8°C, 25°C, and 35°C by multiple linear regression, as described above. The results are also summarized in Table 24.

[0341] As shown in Table 24, the initial (T0) soluble aggregate levels (%HMW) were comparable to or low despite the high protein concentration (within 0.8–1%HMW).

[0342] Analysis of all formulations was performed for up to 12 months at 2–8°C, up to 25°C, and up to 6 months at 35°C. As expected, the results in Table 24 show that the rate of aggregation increased with storage temperature. Within the temperature range of the stability test, the aggregation rate doubled for every 10°C increase in temperature.

[0343] The stability of the Fc polymer molecule (CSL730) at a high protein concentration (160 mg / ml) was, on average, 1.3 times lower at 2–8°C, 25°C, and 35°C compared to the same formulation at a low protein concentration (120 mg / ml).

[0344] For up to 3 months at a fixed molar ratio of approximately 1:420, all stabilizers appeared to perform similarly in stabilizing the Fc polymer molecule (CSL730) at all tested temperatures. This was also confirmed to be true up to 12 months.

[0345] [Table 24]

[0346] [Table 25]

[0347] All formulations from F1 to F6 showed good stability for at least 12 months. At 4°C, all six formulations contained less than 3.5% HMW species after 12 months, and at 25°C, all six formulations contained less than 6.5% HMW species.

[0348] Example 4 Testing of fragmentation behavior (low molecular weight species) at T0 and over time by non-reducing capillary gel electrophoresis (NR-cGE). All formulations (see Table 20) were analyzed by non-reducing cGE using a caliper (NR Caliper) to quantify low molecular weight (LMW) species (i.e., fragmentation) at T0, as described above.

[0349] Table 25 shows the baseline (T0) %LMW for all formulations. As shown in Table 25, the T0 %LMW for all formulations was comparable. The same outcome was observed with LMW measured by the SEC method at T0 (the measured %LMW for all formulations was in the range of 2.6–2.7%).

[0350] The %LMW type of all formulations was monitored over time at different temperatures using the NR-cGE "Caliper" method, initially for 3 months, and then continued for 12 months. The fragmentation rate constant, along with the corresponding standard error (SE), was determined at different storage temperatures by multiple linear regression as described above. The results are summarized in Table 25.

[0351] As expected, the results in Table 25 show an increase in the rate of fragmentation with increasing storage temperature. Small variations in fragmentation (within assay error) occurred in all formulations tested over time at 2–8°C, as evidenced by small changes in %LMW over 12 months. However, for every 10°C increase in storage temperature (from 25°C to 35°C), the rate of fragmentation increased by an average of 3.6–4 times, regardless of protein concentration. Similar outcomes were observed with respect to the rate of fragmentation measured by the SEC method (results not shown). By the SEC method, for every 10°C increase in storage temperature (from 25°C to 35°C), the rate of fragmentation increased by an average of 3.5 times.

[0352] At the same storage temperature, no difference was observed between the stability of the Fc polymer molecule (CSL730) at a high protein concentration (160 mg / ml) and the same formulation at a low protein concentration (120 mg / ml). That is, as a function of protein concentration, there was no significant difference between the stability of the formulation at both 25°C and 35°C. The same outcome was observed for LMW measured by the SEC method (results not shown).

[0353] At a fixed molar ratio of approximately 1:420 for up to 12 months, all stabilizers appeared to perform similarly in stabilizing the FC polymer molecule (CSL730) against fragmentation at 2–8°C, 25°C, and 35°C.

[0354] [Table 26]

[0355] [Table 27]

[0356] Example 5 Testing of acidic and basic species at T0 and over time by cation exchange chromatography (CEX). All formulations (see Table 20) were analyzed by CEX to quantify the % acidic and basic species at T0, as described above.

[0357] The initial (T0) % acidic species for all formulations are shown in Table 26. As shown in Table 26, the % acidic species at T0 were similar for all formulations. Similarly, the % basic species at T0 were also similar for all formulations (3.5–3.7%).

[0358] The % acidity of all formulations was monitored over time at different temperatures using the CEX method (up to 12 months). The rate constant of acidity formation, along with the corresponding standard error (SE), was determined at different storage temperatures by multiple linear regression, as described above. The results are summarized in Table 26.

[0359] As expected, the results in Table 26 show the rate of acidic and basic species formation, which increases with storage temperature. No change in acidic species formation was observed in any of the tested formulations for up to 12 months at 2–8°C. However, for every 10°C increase in storage temperature (from 25°C to 35°C), the rate of acidic species formation increased by an average of 3.1 to 3.4 times.

[0360] The results in Table 26 show that the stability of the Fc polymer molecule (CSL730) at a low protein concentration (120 mg / ml) was slightly better than that of the same formulation at a high protein concentration (160 mg / ml) at a storage temperature of 25°C.

[0361] The results in Table 26 also show that the stability of the Fc polymer molecule (CSL730) was comparable with respect to the formation of acidic species in the presence of sucrose, trehalose, or proline at 25°C, at a fixed molar ratio of approximately 1:420. The results at 35°C are not conclusive due to the limited number of samples available for analysis at that temperature.

[0362] [Table 28]

[0363] No significant differences were observed among the six formulations regarding the percentage of basic species at different time points and temperatures.

Claims

1. 1. An Fc multimeric composition comprising: a) Fc multimer at a concentration between 60 mg / ml and 180 mg / ml; b) a pH value between 4.8 and 6.0; c) a buffer, where the buffer is an acetate buffer, a histidine buffer or a citrate buffer, and d) a stabilizer at a concentration between 200 mM and 450 mM, where the stabilizer is a polyol and / or an amino acid; Including, The Fc multimer composition is composed of three Fc moieties, each Fc moiety being a molecule assembled from two Fc polypeptides capable of binding to an Fc receptor, each Fc polypeptide being a polypeptide chain consisting of a portion of an antibody heavy chain constant domain, and the Fc polypeptides do not contain any portion of an immunoglobulin capable of acting as an antigen recognition region or any complementarity determining region (CDR).

2. 2. The Fc multimer composition of claim 1, comprising an Fc multimer at a concentration between 70 mg / ml and 110 mg / ml.

3. 2. The Fc multimer composition of claim 1, comprising an Fc multimer at a concentration between 110 mg / ml and 180 mg / ml, in particular 120 mg / ml or 160 mg / ml.

4. The Fc multimer composition according to any one of claims 1 to 3, wherein the pH value is between 5.0 and 5.

5.

5. The Fc multimer composition according to any one of claims 1 to 4, wherein the stabiliser is a polyol, in particular a disaccharide, preferably sucrose or trehalose, or sorbitol.

6. The Fc multimer composition according to any one of claims 1 to 5, wherein the molar ratio of Fc multimer to stabilizer is between 1:300 and 1:650, in particular between 1:400 and 1:

425.

7. The Fc multimer composition according to any one of claims 1 to 6, further comprising an antioxidant, in particular methionine.

8. The Fc multimer composition according to any one of claims 1 to 7, further comprising a surfactant, in particular polysorbate 80.

9. An Fc multimer composition comprising the Fc multimer composition of any one of claims 1 to 8 in lyophilized form.

10. The Fc multimeric composition of any one of claims 1 to 9, wherein each Fc moiety comprises two Fc polypeptides, each comprising a hinge region, a CH2 constant domain, and a CH3 constant domain.

11. The Fc multimer composition of claim 10, wherein the Fc multimer comprises two polypeptides having the amino acid sequence of SEQ ID NO:23 and two polypeptides having the amino acid sequence of SEQ ID NO:

24.

12. 12. The Fc multimer composition of any one of claims 1 to 11 for use in the treatment of an autoimmune or inflammatory disease.

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