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 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. [Background technology]

[0002] Immunoglobulins play an important 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 the antigen-binding site, and a constant region that is responsible for the immunoglobulin's effector functions.

[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 major antibody class in exocrine secretions such as saliva, tears, and mucus of the respiratory and intestinal tracts. IgM is by far the largest physical antibody in the human circulatory system and is usually present as a pentamer of basic immunoglobulin units, appearing early in infection.

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

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

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

[0007] Fc receptors are receptors on the outer surface of cells of the immune system, such as human platelets, mast cells, phagocytes such as macrophages and monocytes, granulocytes such as neutrophils, basophils, and eosinophils, and lymphocytes of the innate immune system (natural killer cells) or adaptive immune system (e.g., B cells). Binding of the Fc region of an antibody to an antigen (such as a pathogenic microorganism) via its Fab region usually results in the pathogen's connection to cells of the immune system. As a result, a coordinated response against the pathogen is elicited.

[0008] The complement system is part of the immune system that attacks pathogen cell membranes by forming transmembrane channels that disrupt the pathogen's membrane, which subsequently leads to cell lysis and pathogen death. Activation of the complement system also leads to stimulation of phagocytes, which eliminate foreign and damaging substances, and inflammation, which attracts additional phagocytes.

[0009] Several strategies to replace IVIG / SCIG for the treatment of autoimmune, inflammatory, and neurological disorders are under investigation. One approach is to use only the Fc portion of IgG at high concentrations or to recombinantly assemble several Fc portions into a single molecule, e.g., to increase the binding activity of the Fc portion to Fc receptors. For example, various configurations of multimeric Fc molecules were disclosed in U.S. Patent No. 5,629,493. Advantageous constructs with different configurations of 2 to 10 Fc portions were disclosed (see Momenta's patent application, U.S. Patent No. 5,629,493). The most favorable option was a trivalent human IgG1 Fc molecule produced from two long chains of two Fc polypeptides connected by a flexible peptide linker and two short chains of a single Fc polypeptide. Mutations such as knob-in-hole mutations and electrostatic mutations were introduced to direct the correct assembly of the construct. Another example is a hexameric Fc molecule assembled by addition of the C-terminal 18 amino acid tail piece of IgM to the Fc polypeptide of IgG, as disclosed, for example, in US Pat. Nos. 5,849,999 and 5,849,999.

[0010] For therapeutic use, stable formulations of such Fc multimers are needed. 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 for Fc multimers. Initial attempts to formulate Fc multimers in liquid form were only possible at very low protein concentrations (30 mg / ml). For therapeutic use, more concentrated solutions of these proteins are highly desirable. Stable compositions with suitably high protein concentrations have not been achieved, primarily because, as noted above, Fc multimers 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 [Problem to be solved by the invention]

[0012] An object of the present invention is therefore to provide stable Fc multimer compositions having higher concentrations of Fc multimers, allowing convenient administration of Fc multimers to patients, for example by subcutaneous administration. [Means for solving the problem]

[0013] 1. An Fc multimer composition comprising: a) Fc multimers at a concentration between 60 mg / ml and 180 mg / ml; b) a pH value between 4.8 and 6.0, and c) stabilizer at a concentration between 200 mM and 450 mM The Fc multimer composition is provided, comprising:

[0014] The present invention further relates to Fc multimer compositions, including Fc multimer compositions of the present invention in lyophilized form.

[0015] Further provided are Fc multimer compositions of the present invention for use in treating autoimmune or inflammatory diseases. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows soluble (physical) aggregation (% HMW) over time at 35° C. for formulations 1-12. [Figure 2] 1 is a plot of pooled data of the rate of aggregation at 25° C. and 35° C. as a function of measured pH. [Figure 3]FIG. 1 shows fragmentation (% LMW species) (NE-Caliper) over time at 35° C. for formulations 1 to 12. [Figure 4] FIG. 1 shows a plot of pooled data of the rate of fragmentation (±SE) at 35° C. as a function of measured pH. [Figure 5] FIG. 1 shows the formation of acidic species (CEX) over time at 35° C. for all formulations. [Figure 6] FIG. 1 shows a plot of pooled data of the rate of formation of acidic species at 25° C. and 35° C. as a function of measured pH. [Figure 7] Analysis of particles subvisible to the naked eye. (a) Particle counts (particles / mL) for particles ≥ 10 microns (black bars) and particles ≥ 25 microns (gray bars) for F1, F2, F3, F4, F5, F9, F12, and F17 after 3 months at 5°C and 25°C. (b) Summary of particle counts (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) for F1, F2, F3, F4, F5, F9, F12, and F17 after 3 months at 5°C and 25°C. [Figure 8] FIG. 1 shows the results of the starting (T0) % acidic and % basic species as measured by cation exchange chromatography. [Figure 9] FIG. 1 shows representative chromatograms from RP-HPLC quantifying oxidation of Fc multimeric molecules (CSL730) in formulations. [Figure 10] FIG. 1 shows plots comparing the starting (T0) % short-chain oxidized species, long-chain oxidized species and relative % total oxidation measured by RP-HPLC for all formulations of Fc multimeric molecule (CSL730). [Figure 11] FIG. 1 shows a plot showing the course of % total oxidation, as measured by RP-HPLC, for all formulations of Fc multimeric molecule (CSL730) at 25° C. and 40° C. over up to two months of storage stability. [Figure 12]1 is a diagram of an Fc construct containing three Fc domains formed from four polypeptides like CSL730. The first polypeptide (502) contains one Fc polypeptide containing an amino acid of a different charge at the CH3-CH3 interface, in addition to the wt sequence (506) joined in tandem with a lobe-containing Fc polypeptide (504). The second polypeptide (508) contains an Fc polypeptide containing an amino acid of a different charge at the CH3-CH3 interface, in addition to the wt sequence (512) joined in tandem with another lobe-containing Fc polypeptide (510). The third and fourth polypeptides (514 and 516, respectively) each contain a recess-containing Fc polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0017] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a) Fc multimers at concentrations between 60 mg / ml and 180 mg / ml b) a pH value between 4.8 and 6.0, and c) stabilizer at a concentration between 200 mM and 450 mM The present invention relates to an Fc multimer composition comprising:

[0018] The results of the Examples demonstrate that the Fc multimer compositions described in the present invention are particularly stable. In particular, the Examples demonstrate that even Fc multimer compositions with higher protein concentrations, using the conditions set forth in the claims, are able to prevent protein precipitation and deamidation, resulting in a reduced fragmentation rate.

[0019] The term "Fc multimer" according to the present invention describes a multimer composed of at least two Fc moieties.

[0020] According to the present invention, the terms "Fc portion", "Fc monomer", "Fc fragment" or "Fc domain", used interchangeably herein, describe a molecule assembled from two Fc polypeptides that is capable of binding to an Fc receptor.

[0021] The term "Fc polypeptide" according to the present invention describes a polypeptide chain consisting of part of the heavy chain constant domain of an antibody.

[0022] In particular, the Fc polypeptides of the present invention comprise an antibody constant domain (CH2 and / or CH3) or a functional fragment thereof (e.g., a fragment that (i) dimerizes with another Fc polypeptide to form an Fc portion, and (ii) is capable of binding to an Fc receptor).

[0023] Additionally, the Fc portion can include the hinge region and the constant CH2 and CH3 (and CH4) domains of an immunoglobulin heavy chain constant domain, or any combination of fragments thereof.

[0024] The term "constant domain CH2 / CH3 / CH4" according to the present invention describes the part of the constant domain of the heavy chain of an antibody.

[0025] The term "hinge region" according to the present invention describes a flexible stretch of the polypeptide chain of the constant domain of the heavy chain of an antibody, whose normal function is to connect the Fab and Fc parts of an antibody, or fragments thereof.

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

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

[0028] Typically, the Fc polypeptide according to the present invention comprises an antigen recognition domain. Area and of immunoglobulins that can act as Any Part , any complementarity determining region (CDR) Does not include.

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

[0030] Such changes may be any known change in amino acids known to those of skill in the art, such as amino acid substitutions, additions, or deletions. Furthermore, changes may be conservative or any change that alters 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 alterations are introduced anywhere in the Fc polypeptide, particularly in the hinge region, CH2 or CH3 domain.

[0032] Suitable changes are known to those skilled in the art, and in particular may be knob-into-hole amino acid changes, amino acid changes that result in disulfide bonds, amino acid changes that result in oppositely charged amino acid interactions, as well as the incorporation of peptide linkers in the Fc portion that result in improved interactions of the Fc polypeptide.

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

[0034] In the present invention, engineered recesses and engineered protrusions are used in the production of the Fc moieties described herein.

[0035] The term "engineered recess" refers to the replacement of at least one original amino acid residue, e.g., 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, e.g., in the CH3 constant domain.

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

[0037] Engineered 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, engineered 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 of skill in the art.

[0038] In an Fc moiety according to the invention, two Fc polypeptides may interact such that a first Fc polypeptide may comprise an engineered recess in its CH3 constant domain, while a second Fc polypeptide may comprise an engineered protrusion in its CH3 constant domain.

[0039] In other embodiments, an engineered recess or engineered protrusion in one CH3 constant domain is engineered to better fit the native amino acids of another CH3 constant domain.

[0040] In the present invention, disulfide bond engineering of the CH3 intradomain is further applied to enhance dimer formation, for example, a non-cysteine ​​amino acid of one CH3 constant domain is replaced with a cysteine ​​amino acid to enable disulfide bond formation with a cysteine ​​of another Fc polypeptide, particularly with the CH3 constant domain of an Fc polypeptide.

[0041] Furthermore, the incorporation of one or more peptide linkers can be used to enhance dimer formation. Such peptide linkers typically contain flexible amino acids such as glycine and / or serine. Thus, a first Fc polypeptide is connected to a second Fc polypeptide by a peptide linker, with the N-terminus of the peptide linker being connected to the C-terminus of the first Fc polypeptide through a chemical bond, and the C-terminus of the peptide linker being connected 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, ie, the two portions of the Fc polypeptide are linked via an amino acid chain of two or more glycine molecules.

[0043] When an Fc polypeptide contains one or more alterations, these may be at any position in the molecule.

[0044] An Fc portion according to the present invention is usually assembled from two Fc polypeptides. In particular, an Fc portion according to the present invention is not understood to be a whole antibody.

[0045] The Fc portion is composed of two identical Fc polypeptides. Additionally, the Fc portion is composed of two different Fc polypeptides.

[0046] In the Fc portion, both Fc polypeptides may be mutated as described above, or 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 through any known type of intermolecular and intramolecular forces known to those skilled in the art, such as covalent or noncovalent bonds. Fc polypeptides can interact, for example, through oppositely charged amino acids, which are introduced into their amino acid sequences by amino acid substitution as described above. Furthermore, Fc polypeptides can interact through disulfide bonds resulting from the coupling of two thiol groups from two cysteine ​​residues of different Fc polypeptides. Furthermore, Fc polypeptides can be linked via the peptide linker described above.

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

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

[0050] In another preferred embodiment, the Fc multimer is composed of three Fc moieties.

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

[0052] In some embodiments, the Fc multimer comprises an antigen recognition region, e.g., a variable domain (e.g., a V H , V L , hypervariable regions (HVRs)) or complementarity determining regions (CDRs).

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

[0054] In some embodiments, the complementary dimerization selectivity module promotes selective dimerization of Fc polypeptides. In any of the Fc constructs described herein, the Fc polypeptides can have sequences that differ between two Fc polypeptides (i.e., between an Fc polypeptide and another Fc peptide of the Fc construct), e.g., sequences that differ by 20 amino acids or less (e.g., 15, 10 amino acids or less), e.g., 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 amino acids or less. For example, the complementary dimerization selectivity module of any Fc construct can be selected from the C of one Fc polypeptide and the C of the other Fc polypeptide. H 3. The engineered recess in the antibody constant domain and the C of the Fc polypeptide on the other hand H In some embodiments, the Fc polypeptide sequences of the constructs described herein may be different, as they may comprise engineered lobes of three antibody constant domains, with the engineered recess and engineered lobe positioned to form a lobe-recess pair of Fc polypeptides. H In some embodiments, the Fc construct comprises amino acid modifications in the C3 domain of an Fc polypeptide (one or more Fc polypeptides) for selective dimerization. H In another embodiment, the complementary dimerization selectivity module comprises an amino acid modification in the C3 domain of one Fc polypeptide. Exemplary engineered recesses and protrusions are known in the art. In another embodiment, the complementary dimerization selectivity module comprises an amino acid modification in the C3 domain of one Fc polypeptide. H 3. Negatively charged amino acids engineered (substituted) into the antibody constant domain are inserted into the C of the other Fc polypeptide. H 3 antibody constant domains contain engineered (substituted) positively charged amino acids, with negatively and positively charged amino acids positioned to facilitate 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, only one, two, three, or four Fc polypeptides have the same modification.

[0055] In some embodiments, an Fc multimer comprises at least two Fc monomers connected via a linker. In some embodiments, an Fc multimer comprises at least one linker. The linker may be an amino acid spacer comprising 3 to 200 amino acids (e.g., 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 single, multiple, or repeated motif, such as GS, GGS, GGSG, GGGGS, GGG, or GGGG. In certain embodiments, the linker may comprise GS, GGS, GGSG, GGGGS, GGG, or any of SEQ ID NOs: 34-61. In some embodiments, a linker is used to connect two Fc polypeptides in tandem. In other embodiments, a linker is used to connect two Fc polypeptides in tandem. L and C H1 It connects the antibody constant domains. In other embodiments, the linker may contain amino acids other than glycine and serine.

[0056] In some embodiments described herein, an Fc multimer can comprise a polypeptide comprising SEQ ID NO: 1-31 or SEQ ID NO: 1-31 with up to 10 (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1) single amino acid alterations (e.g., substitutions, e.g., conservative substitutions). In some embodiments, the Fc polypeptides of an Fc domain of a construct can have sequences that differ between two Fc polypeptides (i.e., between an Fc polypeptide of an Fc construct and another Fc polypeptide), e.g., sequences that differ by no more than 20 amino acids (e.g., no more than 15, 10 amino acids), e.g., no more than 20, 15, 10, 8, 7, 6, 5, 4, 3, or 2 amino acids.

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

[0058] In one embodiment, the Fc multimers of the present invention are disclosed in WO2015 / 168643A2, WO2017 / 205436A1, WO2017 / 205434A1, and WO2018 / 129255A1, the entire contents of which are incorporated herein by reference. Preferably, the Fc multimer comprises an Fc construct having 2 to 10 Fc domains, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains. In some embodiments, the Fc multimer comprises three Fc domains.

[0059] In one aspect, the disclosure features an Fc construct including four polypeptides forming three Fc domains. The first polypeptide has the formula ALB, where A includes a first Fc polypeptide; L is a linker; and B includes a second Fc polypeptide. The second polypeptide has the formula A'-L'-B', where A' includes a third Fc polypeptide; L' is a linker; and B' includes a fourth Fc polypeptide. The third polypeptide includes a fifth Fc polypeptide, and the fourth polypeptide includes a sixth Fc polypeptide. In this aspect, 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 aspect, A and A' each comprise a dimerization selectivity module that promotes dimerization between these Fc polypeptides. In other embodiments, B and the fifth Fc polypeptide each comprise a dimerization selectivity module that promotes dimerization between these Fc polypeptides. In yet other embodiments, B' and the sixth Fc polypeptide each comprise a dimerization selectivity module that promotes dimerization between these Fc polypeptides.

[0061] In some embodiments of this aspect, one or more of A, B, A', B', the third polypeptide, and the fourth polypeptide consist of an Fc polypeptide, hi some embodiments, each of A, B, A', B', the third polypeptide, and the fourth polypeptide consist of an Fc polypeptide.

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

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

[0064] In some embodiments of this aspect, each of L and L' comprises at least 4, 8, 12, 14, 16, 18, or 20 glycines. In some embodiments, each of L and L' comprises between 4 and 30, between 8 and 30, or between 12 and 30 glycines. In some embodiments of this aspect, each of L and L' comprises, consists of, or consists essentially of GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 61).

[0065] In some embodiments, the Fc construct further comprises a heterologous moiety, eg, a peptide, eg, an albumin binding peptide, connected 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 comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 24, and the third and fourth polypeptides comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 23. In some embodiments, the first and second polypeptides comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 24, and the third and fourth polypeptides comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 23 with an asparagine to glutamine substitution at position 162. In some embodiments, the Fc multimer in the composition 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. In some embodiments, the Fc multimer in the composition comprises two polypeptides having the amino acid sequence of SEQ ID NO: 23 with an asparagine to glutamine substitution at position 162 and two polypeptides having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the Fc multimer 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, wherein about 12% of the polypeptides have an asparagine to glutamine substitution at position 162. In some embodiments of the present disclosure, each of the first and second polypeptides comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 24 with 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, consist of, or consist essentially of the sequence of SEQ ID NO: 23 with 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 comprise, consist of, or consist essentially of the sequence of SEQ ID NO:29, and the third and fourth polypeptides comprise, consist of, or consist essentially of the sequence of SEQ ID NO:28.In some embodiments of the present disclosure, each of the first and second polypeptides comprises, consists of, or consists essentially of the sequence of SEQ ID NO: 29 with 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, consist of, or consist essentially of the sequence of SEQ ID NO: 28 with 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 disclosure, the first and third Fc polypeptides each comprise a complementary dimerization selectivity module that promotes dimerization between the first Fc polypeptide and the third Fc polypeptide, and the second and fourth Fc polypeptides each comprise a complementary dimerization selectivity module that promotes dimerization between the second Fc polypeptide and the fourth Fc polypeptide. In some embodiments, the complementary dimerization selectivity module of each of the first and second Fc polypeptides comprises an engineered protrusion, and the complementary dimerization selectivity module of each of the third and fourth Fc polypeptides comprises an engineered recess.

[0069] In some embodiments, one or more Fc domain monomers comprise an IgG hinge region or portion thereof, an IgG C H 2 antibody constant domains, and IgG C H In some embodiments, each of the Fc polypeptides comprises an IgG hinge region or portion thereof, an IgG C region, an IgG D region, an IgG E region, an IgG H region, an IgG I ... H 2 antibody constant domains, and IgG C H In some embodiments, each of the Fc polypeptides comprises 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 previous two aspects of the disclosure, an N-terminal aspartic acid of one or more of the first, second, third, and fourth polypeptides is mutated to glutamine, hi some embodiments, an N-terminal aspartic acid of each of the first, second, third, and fourth polypeptides is mutated to glutamine.

[0071] In some embodiments, one or more of the first, second, third, and fourth polypeptides lack a C-terminal lysine, hi some embodiments, each of the first, second, third, and fourth polypeptides lacks a 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 comprises an amino acid modification that alters one or more of: (i) binding affinity to one or more Fc receptors, (ii) effector function, (iii) level of Fc domain sulfation, (iv) half-life, (v) protease resistance, (vi) Fc domain stability, and / or (vii) susceptibility to degradation. In some embodiments, the Fc domain comprises an amino acid modification, e.g., S267E / L328F, that alters binding affinity to one or more Fc receptors. In some embodiments, the Fc receptor is FcγRIIb. In some cases, the modifications described herein increase affinity for the FcγRIIb receptor. In some cases, the S267E / L328F modification increases binding affinity to FcγRIIb. In some embodiments, the Fc domain comprises an amino acid modification, e.g., 241F, 243F, 246K, 260T, or 301R, that alters the level of Fc domain sulfation. In some embodiments, the Fc domain comprises amino acid modifications that alter protease resistance, e.g., selected from the following set: 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 comprises an amino acid modification that alters the susceptibility 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] 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 Fc polypeptide and the fifth Fc polypeptide combine to form a first Fc domain, the second Fc polypeptide and the fourth Fc polypeptide combine to form a second Fc domain, and the third Fc polypeptide and the sixth Fc polypeptide combine to form a third Fc domain, and wherein at least one Fc domain comprises an amino acid modification at position 1253 (e.g., a single amino acid modification at position 1253).

[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 comprises an amino acid modification at position 1253. In some cases, one or both of the first and fifth Fc polypeptides comprise an amino acid substitution at position 1253. In some embodiments, the second Fc domain comprises an amino acid modification at position 1253. In some embodiments, one or both of the second and fourth Fc polypeptides comprise an amino acid substitution at position 1253. In some embodiments, the third Fc domain comprises an amino acid modification at position 1253. In some embodiments, one or both of the third and sixth Fc polypeptides comprise an amino acid substitution at position 1253. 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] 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 Fc polypeptide and the fifth Fc polypeptide combine to form a first Fc domain, the second Fc polypeptide and the fourth Fc polypeptide combine to form a second Fc domain, and the third Fc polypeptide and the sixth Fc polypeptide combine to form a third Fc domain, and wherein at least one Fc domain comprises an amino acid modification at position R292 (e.g., a single amino acid modification).

[0077] In some embodiments, the first Fc domain comprises an amino acid modification at position R292. In some embodiments, one or both of the first and fifth Fc polypeptides comprise an amino acid substitution at position R292. In some embodiments, the second Fc domain comprises an amino acid modification at position R292. In some embodiments, one or both of the second and fourth Fc polypeptides comprise an amino acid substitution at position R292. In some embodiments, the third Fc domain comprises an amino acid modification at position R292. In some embodiments, one or both of the third and sixth Fc polypeptides comprise an amino acid substitution at position R292. In some embodiments, each of the first, second, and third Fc domains comprises an amino acid modification (e.g., substitution) at position R292P (i.e., each Fc monomer has an R292P modification, e.g., compared to SEQ ID NO: 23). In some embodiments, one or both of the first and fifth Fc polypeptides comprises the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides comprises the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides comprises 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 comprises the amino acid modification (e.g., substitution) I253A, and each of the first, second, and third Fc domains comprises the amino acid modification (e.g., substitution) R292P. In some embodiments, one or both of the first and fifth Fc polypeptides comprise the amino acid substitution 1253A, one or both of the third and sixth Fc polypeptides comprise the amino acid substitution 1253A, one or both of the first and fifth Fc polypeptides comprise the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides comprise the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides comprise the amino acid substitution R292P. In some embodiments, each of the first, second, and third Fc domains comprises the amino acid modifications (e.g., substitutions) 1253A and R292P. In some embodiments, one or both of the first and fifth Fc polypeptides comprise the amino acid substitution I253A, one or both of the second and fourth Fc polypeptides comprise the amino acid substitution I253A, and one or both of the third and sixth Fc polypeptides comprise the amino acid substitution I253A, one or both of the first and fifth Fc polypeptides comprise the amino acid substitution R292P, one or both of the second and fourth Fc polypeptides comprise the amino acid substitution R292P, and one or both of the third and sixth Fc polypeptides comprise the amino acid substitution R292P.

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

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

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

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

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

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

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

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

[0087] In some embodiments, the Fc constructs described herein do not contain an antigen recognition region, such as a variable domain or a complementarity-determining region (CDR). In some embodiments, the Fc construct (or the 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 contain an additional domain (e.g., an IgM tail or an IgA tail) that facilitates the association of the two polypeptides. In other embodiments, a covalent bond (e.g., a disulfide bridge) exists only between the two Fc polypeptides that join to form the Fc domain. In other embodiments, the Fc construct does not contain a covalent bond (e.g., a disulfide bridge) between the Fc domains. In yet other embodiments, the Fc construct provides sufficient structural flexibility so that all or substantially all Fc domains of the Fc construct can simultaneously interact with Fc receptors on the cell surface. In one embodiment, the Fc polypeptides differ in primary sequence from each other, either from wild-type or with a dimerization selectivity module.

[0088] In another aspect, the disclosure features compositions and methods for promoting selective dimerization of Fc polypeptides. The disclosure provides compositions and methods for promoting selective dimerization of Fc polypeptides, including an Fc domain, wherein two Fc polypeptides of the Fc domain are C H The present disclosure provides a method for identifying C3 antibody constant domains comprising the same mutations at at least two positions within the ring of charged residues at the interface between the C3 antibody constant domains. H Also included is a method for making such an Fc domain, comprising introducing complementary dimerization selectivity modules with identical mutations in two Fc polypeptide sequences at at least two positions within the 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 3Antibody constant domain is another C HWhen combined with three antibody constant domains, these charged residues pair with residues of the opposite charge. By reversing the charge of both members of two or more pairs of complementary residues, the mutant Fc polypeptide maintains complementarity to Fc polypeptides of the same mutant sequence but has lower complementarity to 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 comprises an Fc polypeptide comprising a quadruple mutation combining any pair of double mutations, for example, K409D / D399K / E357K / K370E.

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

[0090] It is understood that in any Fc construct described herein, the order of the Fc polypeptides can be interchanged. For example, in a polypeptide having the formula ALB, the carboxy terminus of A is joined to the amino terminus of L, which is then joined at its carboxy terminus to the amino terminus of B. Alternatively, the carboxy terminus of B is joined to the amino terminus of L, which is then joined at its carboxy terminus to the amino terminus of C. Both of these structures are encompassed by the formula ALB.

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

[0092] As described in further detail herein, the present disclosure features substantially homogenous compositions containing Fc constructs that all have the same number of Fc domains, as well as methods for preparing such substantially homogenous compositions.

[0093] The Fc constructs of the present disclosure can be included in pharmaceutical compositions comprising a substantially homogenous population (e.g., at least 85%, 90%, 95%, 98%, or 99% homogeneity) of Fc constructs having 2-10 Fc domains (e.g., 2-8 Fc domains, 2-6 Fc domains, 2-4 Fc domains, 2-3 Fc domains, 3-5 Fc domains, or 5-10 Fc domains), e.g., constructs having 2, 3, 4, 5, 6, 7, 8, 9, or 10 Fc domains, such as those described herein. As a result, pharmaceutical compositions can be produced that do not have substantial aggregation or unwanted multimerization of Fc constructs.

[0094] Furthermore, the Fc multimer is composed of six Fc moieties, which are IgG Fc moieties, and the addition of an 18-amino acid tail piece at 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, and each Fc polypeptide contains an IgG Fc polypeptide and an IgM tail piece.

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

[0096] For example, in one embodiment, the Fc polypeptide comprises an IgG1 hinge region, an IgG1 Fc region, and an IgM tailpiece, and does not comprise a Fab polypeptide. In a preferred embodiment, the Fc polypeptide is SEQ ID NO: 32 with up to five conservative amino acid changes.

[0097] In a preferred embodiment, the Fc polypeptide comprises an IgG1 hinge region, an IgG1 Fc region, and an IgM tailpiece, wherein the IgG1 Fc region has a cysteine ​​instead of leucine at position 309 (according to EU numbering), the Fc polypeptide does not comprise a Fab polypeptide, and the Fc polypeptide is SEQ ID NO: 33. In one embodiment, the Fc polypeptide is SEQ ID NO: 33 with up to five conservative amino acid changes.

[0098] Generally, an Fc multimer is composed of identical Fc portions. Furthermore, an Fc multimer is composed of Fc portions that are all different from one another or that are partially identical and partially different from one another in any combination.

[0099] In an Fc multimer, all Fc portions may be mutated, or none of the Fc portions may be mutated, or any number of the Fc portions may be mutated. In particular, the Fc portion according to the present invention is not understood to be a whole antibody.

[0100] The Fc polypeptide, Fc portion, or Fc multimer can be produced by any method for antibody production, Fc portion production, or protein production known to those skilled in the art. Methods for producing Fc polypeptides, Fc portions, or Fc multimers are generally well known to those skilled in the art. For example, the Fc polypeptide, Fc portion, or Fc multimer can be produced with the help of mammalian cells, bacterial cells, or insect cells after introducing a nucleic acid encoding the protein into the mammalian cells, bacterial cells, or insect cells. The Fc polypeptide, Fc portion, or Fc multimer can then be isolated from the mammalian cells, bacterial cells, or insect cells or recovered from the supernatant.

[0101] The Fc multimer compositions according to the present invention comprise Fc multimers at a concentration between 60 mg / ml and 180 mg / ml.

[0102] The Fc multimer compositions according to the present invention particularly comprise Fc multimers at a concentration between 70 mg / ml and 160 mg / ml, more particularly between 80 mg / ml and 120 mg / ml, even more particularly between 90 mg / ml and 110 mg / ml, and most particularly between 95 mg / ml and 105 mg / ml.

[0103] Furthermore, the Fc multimer compositions according to the present invention particularly comprise Fc multimers at a concentration between 70 mg / ml and 160 mg / ml, more particularly between 70 mg / ml and 140 mg / ml, even more particularly between 70 mg / ml and 120 mg / ml, and most particularly between 70 mg / ml and 110 mg / ml.

[0104] In a preferred embodiment, the Fc multimer composition according to the present invention comprises Fc multimers at concentrations of 70 mg / ml and 110 mg / ml.

[0105] Furthermore, the Fc multimer compositions according to the present invention particularly comprise Fc multimers at a concentration between 90 mg / ml and 180 mg / ml, more particularly between 110 mg / ml and 180 mg / ml, even more particularly between 115 mg / ml and 160 mg / ml, and most particularly between 120 mg / ml and 160 mg / ml.

[0106] In another preferred embodiment, the Fc multimer composition according to the invention comprises Fc multimers at a concentration between 110 mg / ml and 180 mg / ml, in particular at 120 mg / ml or 160 mg / ml.

[0107] The protein concentration of the Fc multimer composition of the present invention can be measured by any suitable technique known to those skilled in the art. For example, the protein concentration of the Fc multimer composition of the present invention can be measured by absorption spectroscopy, colorimetry (e.g., Bradford, Biuret, Bicaminic Acid Assay (BCA), or Lowry), gravimetric analysis (e.g., Kjeldahl), quantitative amino acid analysis (e.g., total hydrolysis, end-group analysis, and Edman degradation), or refractometry. Preferably, UV spectroscopy measuring the absorption at 280 nm was used to measure the protein concentration of the Fc multimer composition of the present invention.

[0108] The Fc multimer compositions according to the present invention further comprise a pH value between 4.8 and 6.0.

[0109] In particular, the Fc multimer compositions according to the present invention comprise a pH value between 4.9 and 5.8, more particularly, the Fc multimer compositions according to the present invention comprise a pH value between 5.0 and 5.6, and most particularly, the Fc multimer compositions according to the present invention comprise a pH value between 5.0 and 5.5.

[0110] In a preferred embodiment, the Fc multimer composition according to the present invention comprises a pH value between 5.0 and 5.5.

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

[0112] The Fc multimer compositions according to the present invention further comprise a stabilizer at a concentration between 200 mM and 450 mM.

[0113] The term "stabilizer" according to the present invention may be any substance that prevents the degradation of any component of the composition of the present invention. In particular, the stabilizer is a substance that prevents the degradation and / or aggregation of the Fc multimers and / or Fc portions of the composition of the present invention.

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

[0115] In particular, the stabilizer used in the present invention may be a polyol and / or an amino acid.

[0116] The polyols used as stabilizers in the compositions of the present invention may be organic molecules 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 polyols may be linear or branched. Furthermore, the polyols may be cyclic molecules containing one, two, or more rings. The polyols may contain at least two, three, four, five, six, seven, or eight hydroxy groups. Furthermore, the polyols may be aliphatic, alicyclic, or aromatic.

[0117] Suitable polyols for use 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 stabilizer is a disaccharide such as sucrose or trehalose, and / or sorbitol.

[0118] Suitable amino acids that can be used 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 can be used as stabilizers in the compositions of the present invention.More particularly, proline or arginine can be used as stabilizers in the compositions of the present invention.

[0119] In a preferred embodiment, the Fc multimer compositions according to the present invention comprise a polyol, in particular a disaccharide, preferably sucrose or trehalose, or a reducing sugar such as sorbitol, as a stabilizer.

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

[0121] The compositions according to the present invention may comprise 50 mM to 500 mM of stabilizer, in particular 100 mM to 400 mM of stabilizer, more in particular 200 mM to 300 mM of stabilizer, most in particular 225 mM to 275 mM of stabilizer.

[0122] Compositions according to the invention may comprise 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 may be used, for example at least 100 mM sucrose, or 100 mM to 250 mM sucrose.

[0123] The composition according to the present invention may comprise 100 mM to 500 mM trehalose, in particular 150 mM to 400 mM trehalose, more in particular 200 mM to 300 mM trehalose.

[0124] The composition according to the present invention may comprise 50 mM to 500 mM sorbitol, in particular 100 mM to 400 mM sorbitol, more in particular 200 mM to 300 mM sorbitol, most in particular 225 mM to 275 mM sorbitol.

[0125] In the Fc multimer compositions according to the present invention, the molar ratio of Fc multimer to 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 a preferred embodiment, the molar ratio of Fc multimer to stabilizer in the Fc multimer composition described herein is between 1:300 and 1:650, particularly between 1:400 and 1:425. Preferably, the Fc multimer composition of this embodiment contains 10% or less aggregates (as determined by SEC) when stored at 2-8°C for 12 months, more preferably 7% or less, even more preferably 5% or less, and most preferably 3.5% or less. Preferably, the Fc multimer composition of this embodiment contains 15% or less aggregates, more preferably 10% or less, even more preferably 8% or less, and most preferably 6.5% or less when stored at 25°C.

[0127] Additionally, compositions according to the present invention may or may not contain a buffer.

[0128] In a preferred embodiment, the Fc multimer composition according to the present invention comprises a buffer.

[0129] The term "buffer" according to the present invention describes a mixture of a weak acid and its conjugate base, or vice versa, in its buffering region that maintains its pH at a nearly constant value when additional acid or base is added to it.

[0130] The Fc multimer 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. Generally, any buffer suitable for pharmaceutical applications, particularly suitable for administration to the body of a subject such as a human patient, may be used. Examples of suitable buffers in 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 multimer composition according to the present invention comprises a buffer solution, which is an acetate buffer solution, a histidine buffer solution, or a citrate buffer solution. In a preferred embodiment, the Fc multimer composition according to the present invention comprises a histidine buffer solution.

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

[0133] Examples of suitable antioxidants are ascorbic acid, vitamin E, reduced glutathione (GSH), and / or methionine. In particular, the Fc multimer compositions according to the present invention may contain methionine.

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

[0135] In particular, the Fc multimer composition may comprise 0.01 mM to 100 mM methionine, particularly 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 multimer composition according to the present invention further comprises an antioxidant, in particular methionine.

[0137] The composition according to the present invention may further comprise a surfactant.The term "surfactant" according to the present invention describes 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.Therefore, when dissolved in water, the surfactant reduces the advancing contact angle and helps to replace the air phase on the surface, which can then be replaced by the liquid phase.

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

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

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

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

[0142] Generally, the composition of the present invention may contain one or more surfactants.When the composition contains more than one surfactant, they may belong to the same or different surfactant subclasses.Furthermore, when the composition contains more than one surfactant, they may be any mixture of the above surfactants.

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

[0144] In a preferred embodiment, the Fc multimer composition according to the present invention further comprises a surfactant, in particular polysorbate 80.

[0145] The Fc multimer compositions according to the present invention may further comprise conventional pharmaceutical additives and adjuvants, excipients, or diluents, such as other substances that do not deleteriously react with the active compounds, including, but not limited to, water, salt, gelatin of any origin, vegetable gum, lignosulfonate, talc, sugar, starch, gum arabic, vegetable oil, polyalkylene glycol, flavorings, preservatives, emulsifiers, lubricants, coloring agents, humectants, fillers, etc. The Fc multimer compositions may further comprise enzymes, such as protease inhibitors.

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

[0147] Optionally, the suspension may also contain additional suitable agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0148] In one embodiment of the present invention, the Fc multimer composition according to the present invention may comprise 70 mg / ml of Fc multimer, in particular CSL730, L-histidine buffer, sodium chloride and L-proline, polysorbate 80, and water for injection.

[0149] The Fc multimer composition may be sterilized or processed by any method known to those skilled in the art to render the composition suitable for preservation for storage.

[0150] In a second aspect, the present invention relates to an Fc multimer composition comprising an Fc multimer composition of the present invention in lyophilized form.

[0151] All embodiments described above regarding the Fc multimer composition according to the first aspect are also applicable to the second aspect.

[0152] The term "freeze drying" according to the present invention means any low temperature dehydration method that involves freezing the product, reducing the pressure and removing the ice by sublimation.

[0153] The Fc multimer composition is lyophilized by any lyophilization technique known to those skilled in the art. Lyophilization can be carried out by any means and at any temperature. Preferably, lyophilization is carried out at room temperature (RT).

[0154] The Fc multimer composition may be pre-treated prior to lyophilization, for example, including a step to concentrate the Fc multimer composition.

[0155] Freeze drying can involve a first stage of primary drying. During the primary drying stage, the pressure is reduced (to the range of a few millibars) and sufficient heat is supplied to the substance to sublimate the ice. The amount of heat required is calculated using the latent heat of sublimation of the sublimating molecules. During this first drying stage, approximately 95% of the water in the substance is sublimated. If too much heat is applied, this stage can be slow (industrially it can take several days) because the structure of the substance will be altered.

[0156] In the primary drying stage, pressure is controlled by the application of a partial vacuum. Vacuum typically accelerates sublimation and is useful as a deliberate drying method. Additionally, cooled condenser chambers and / or condenser plates can provide a surface for re-condensation and solidification of water vapor.

[0157] Freeze-drying may include a second stage of secondary drying, in which the temperature is elevated above the primary drying stage and may be above 0°C. Usually, the pressure is also reduced in this stage to aid desorption (typically in the microbar range, or fractions of a Pascal). However, some products may benefit from increased pressure as well. After lyophilization, buffer components may remain in the form of dry salts.

[0158] Furthermore, after the freeze-drying process, the material is sealed, such as in a glass vial. Generally, the resulting powder is reconstituted by adding a suitable solvent before use. The solution may be an aqueous solution, such as a buffered saline solution. Preferably, freeze-drying is carried out until (most of) the liquid is removed. At the end of the freeze-drying process, 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 lyophilisates described herein may be substantially pure and / or sterile.

[0160] In a preferred embodiment, in the lyophilized Fc multimer composition of the present invention, the Fc multimer is composed of 3 to 10 Fc moieties.

[0161] In a more preferred embodiment, in the lyophilized form of the Fc multimer composition of the present invention, the Fc multimer is composed of three Fc moieties.

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

[0163] The term "autoimmune disease" according to the present invention describes a condition resulting from an abnormal immune response to normal body sites 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 administering an Fc portion or an Fc multimer. Generally, the Fc multimer compositions of the present invention are used in the treatment of autoimmune diseases at any body site.

[0165] Examples of autoimmune diseases treated by the Fc multimer compositions 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), myasthenia gravis, ulcerative colitis, immune complex-mediated nephropathy, scleroderma, dermatomyositis, neuromyelitis optica, Sjogren's syndrome, systemic vasculitis, or systemic lupus erythematosus.

[0166] The term "inflammatory disease" according to the present 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 usually involves immune cells, blood vessels, and molecular mediators.

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

[0168] Furthermore, all of the above embodiments relating to the Fc multimer compositions described in the first aspect also apply to the third aspect.

[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, for example, 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 subcutaneous administration thereof.

[0170] Furthermore, the use of the Fc multimer composition of the present invention in the treatment of autoimmune or inflammatory diseases involves the concomitant use of an additional immunoglobulin or portion thereof, a nonsteroidal anti-inflammatory drug (NSAID), and / or an immunosuppressant, wherein the Fc multimer composition and the additional immunoglobulin or portion thereof, the nonsteroidal anti-inflammatory drug (NSAID), and / or the immunosuppressant are administered together in a single administration or separately in two or more administrations.

[0171] The present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "encompass" are to be interpreted inclusively rather than exclusively.

[0172] Unless otherwise defined, all technical and scientific terms and acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein.

[0173] The present invention is further illustrated by the following figures and examples, which are intended to illustrate but not limit the present invention, and from which additional features, embodiments, and advantages may be read. As such, the specific modifications described are not to be construed as limiting the scope of the invention. Various equivalents, changes, and modifications can be made by those skilled in the art without departing from the scope of the invention, and therefore, such equivalent embodiments will be understood to be included herein. [Example]

[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 multimeric molecules (CSL730), a CSF730 formulation with a protein concentration of 100 mg / mL was prepared. and examined protein stability.

[0175] CSL730 is a trivalent Fc multimer consisting of two long chains of SEQ ID NO: 24 and two short chains of SEQ ID NO: 23, produced as described in WO2015 / 168643A2, which results in a trivalent Fc multimer as shown in Figure 12.

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

[0177] [Table 1]

[0178] Preparation of formulations: The formulations were prepared with CSL730 (recombinant Fc multimer 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 formulations were dialyzed against approximately 1 L of buffer for approximately 5 hours, then replaced 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 dialysis: 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 (unbuffered)

[0179] After dialysis, the protein concentration of each sample was measured. If samples were found to be too dilute, they were concentrated by centrifugation using Amicon tubes (30 kDa cutoff) aiming for 100±10 mg / mL. The protein concentration was then confirmed.

[0180] Filling and finishing 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 labeled 2 ml glass vials. The vials were 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. Acetate, histidine, and citrate buffers were used at different pH levels to achieve good buffer performance within the pH range of 4.5 to 7. No surfactants were added to the formulations in this study. 50 mM sucrose was added to all formulations as a stabilizer to provide a minimal level of stability. Note that sucrose does not affect ionic strength. One buffer-free formulation was prepared because highly concentrated proteins are known to be self-buffering. The pH of the buffer-free solution will depend on the protein itself.

[0182] [Table 2]

[0183] After filling, the filled formulations were placed in stability chambers at 2-8°C, 25°C, or 35°C. Samples were analyzed immediately after the filling / finishing step (for "t0" / t=0 analysis) and at predetermined subsequent time points up to 4 weeks. To compare the degradation kinetics between different formulations, rate constants (with standard errors) for the different pathways of degradation were determined 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: The formulations were analyzed at different time points using stability-indicating methods, mainly: size exclusion chromatography (SEC), cation exchange chromatography (CEX), and capillary gel electrophoresis (CGE) (Caliper). Additionally, the pH, appearance (color, turbidity, and visible particles) of the formulations were monitored at different time points.

[0185] The methods used in this study, the purpose of each method, and the analyses used at each analysis time point are summarized in Table 3.

[0186] [Table 3]

[0187] The analysis method is explained below:

[0188] Visual Inspection: Visual inspection was performed at an inspection station equipped with a white and black background and fluorescent lighting. The formulation in the vial was gently stirred to avoid foaming, and then inspected for color, clarity, and the presence of visible particles. 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 by using A280 / UV measurements of undiluted formulations on an IMPLEN P360 Nanophotometer. Measurements were performed in triplicate per formulation and the mean of the measurements was calculated.

[0191] Size-exclusion chromatography (SEC)-high-performance liquid chromatography (HPLC): SEC-HPLC was used to determine the protein aggregation profile of the formulations. Samples were analyzed using a Dionex system (Ultimate 3000) equipped with an Acquity BEH200 column (Waters, 4.6 x 150 mm). Samples were diluted to 10 g / L in the appropriate buffer, 3.0 μL was injected, and separation was performed under isocratic conditions at a flow rate of 0.3 mL / min. The mobile phase consisted of BET buffer (pH 6.5) with a runtime of 15 min. Intact protein was detected at 280 nm with a retention time of approximately 3.5 min. Monomeric, high molecular weight (HMWS, aggregates), and low molecular weight (LMWS, fragments) species 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 proteinaceous acidic, major, and basic species. Samples were analyzed using a Dionex system (Ultimate 3000) equipped with a Propack WCX-10 column (Thermo Fisher, 4 × 250 mm). Samples were diluted to 10 g / L in the appropriate buffer, and separation was performed using a 3.0 μL injection volume with a 0.7 mL / min gradient. Briefly, two aqueous buffers (imidazole, piperazine, and TRIS-based) at pH 5 and pH 10 were changed over a 55-minute period. Species were detected at 280 nm, identified against reference standards, and reported as relative area percentages 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: separation, staining, destaining, and detection. Denatured proteins were loaded directly onto the chip from a microtiter plate through a capillary sipper. The sample was 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-reduced samples, the protein solution was diluted to 1 g / L with buffer and Milliq water. For reduced samples, the protein solution was diluted with 1 M DTT. Denaturation occurred at 40°C for 20 minutes for non-reduced samples and at 80°C for 15 minutes for reduced samples. Results are reported as the relative area percentage of intact LMWS and HMWS for non-reduced samples. For reduced samples, long- and short-chain fractions were examined.

[0194] Example 1 Visual inspection at t=0 All formulations were evaluated for appearance immediately prior to filling. Therefore, all filled vials of all formulations were evaluated for appearance after filling / final processing.

[0195] Due to the high protein concentration, all formulations exhibited a slightly opalescent brownish-yellow color. The opalescence was observed to increase with pH. CSL730 in formulations at pH 6.5 and pH 7 precipitated with a stronger precipitation than seen at pH 7. It is well known that higher pH can be used to precipitate Fc fragments. The precipitated fragments were later recovered by solubilization in low pH buffers. This was confirmed by the precipitated portion of the recombinant Fc multimer (data not shown). However, the impact of this high pH exposure on storage stability is not well understood.

[0196] All formulations (including precipitated formulations) are filtered before filling into vials and retested after stoppering and crimping. All formulations have a brownish-yellow color, are slightly opalescent, and are free of visible particles (including the pH 6.5 and pH 7.0 formulations). Gelling 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 lower pH values ​​down to pH 4.5 were more suitable as the lower pH did not result in protein precipitation.

[0198] Example 2 pH and protein concentration measurements at t=0 pH and protein concentration measurements were performed at t=0 for all formulations 1-12 (see Table 2). Differences from target values ​​were also assessed. Results are summarized in Table 4.

[0199] pH measurements showed that the measured pH values ​​were within ±0.2 pH units for most formulations compared to the target pH values, except for 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] With regard to measured protein concentration, most formulations were within the target concentration of 100 + / - 10 mg / mL. The exceptions were the acetate formulations at target pH 4.5 and 5.0. These samples were over-diluted by mistake, which is considered test deviation. On the other hand, the histidine formulations at pH 6.5 and 7.0 had low concentrations as a result of precipitation.

[0201] The results show that the pH values ​​of the formulations were in most cases as expected: with the exception of the acetic acid formulations, a significant decrease in protein concentration at pH ≥ 6.5 is associated with significant precipitation of the protein under these conditions.

[0202] [Table 4]

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

[0204] The soluble aggregate profiles of all formulations showed a relatively low % HMW (total <1.5%) for all formulations, with a general trend indicating that increasing pH was 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 ≤ 1.5%. This indicates that pH ≥ 6.5 is not suitable for formulating Fc multimeric molecules at high protein concentrations, but may be suitable for formulating recombinant Fc multimeric 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 Caliper (NR Caliper) for quantitation of low molecular weight species (fragments) at t=0, as described above.

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

[0207] Example 5 Acidic and basic species profile at t=0 by cation exchange chromatography (CEX) All formulations were analyzed by cation exchange chromatography (CEX) for quantification of acidic and basic species at t=0 as described above.

[0208] All formulations showed similar levels of % acidic species except for formulations 7 and 8. The percentage of acidic species was low in formulation 7 (52%) and lowest in formulation 8 (45%).

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

[0210] All formulations showed similar levels of % basic species except for formulations 7 and 8. Compared to all formulations, the percentage of basic species was lower in formulations 7 and 8 (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 indicates that there may be a threshold pH above 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] Overview of Examples 1 to 5 Based on the analyses performed during processing and immediately after the fill / finish step (T=0), it can be concluded that formulations at pH ≥ 6.5 are not suitable for the formulation of Fc multimeric molecules at high concentrations (≥ 100 mg / mL).

[0214] Nevertheless, all formulations were made stable across a range of pH values ​​for all formulations, covering degradation kinetics.

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

[0216] As above, two examiners (Insp1 and Insp2) inspected the appearance at each time point and temperature, and in most cases there was general alignment between the examiners.

[0217] At all storage temperatures, no changes were observed in the color and clarity of most formulations. The color of all formulations remained slightly brownish yellow, while the clarity remained slightly milky white. Formulations at higher storage temperatures began to show visible particles, but the most significant particle formation occurred at pH ≥ 6.5. Interestingly, formulations with salt showed no visible particles compared to the same formulations at pH 6.

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

[0219] [Table 5]

[0220] Example 7 pH and protein concentration over time The 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 error, the results showed minimal shifts in pH and protein concentration at 2-8°C, 25°C, and 35°C for all formulations over up to 4 weeks of storage.

[0222] Example 8 Relationship between high molecular weight (HMW) formation and pH / buffer / salt over time (size exclusion chromatography) The %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 species over time for formulations stored at 35°C is shown in Figure 1. Figure 1 shows an initial fast increase in %HMW species followed by a slower increase in species over time. The same trend was observed for formulations stored at 25°C.

[0223] The % change in aggregated species (% HMW) after 4 weeks of storage at 2-8°C is shown in Table 6. Aggregation rate constants, along with corresponding standard errors (SE), were determined for all formulations (from three time points) at 25°C and 35°C by multiple linear regression using Graphpad Prism software. The results are summarized in Table 6.

[0224] The results in Table 6 show that the rate of aggregation increased with temperature and pH. A faster rate of aggregation occurred at pH ≥ 6.0. The trend was more pronounced at 35°C. The formulation in histidine buffer at pH 6.9 (Formulation 8) appears to be the most stable. However, this is an artifact of the very low protein concentration (18 mg / mL) in this formulation after protein precipitation, compared to the higher protein concentrations of all other formulations (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 buffered formulation at equivalent pH (Formulation 5). The addition of salt (resulting in higher ionic strength) had no significant effect on improving aggregation behavior.

[0226] The aggregation trends are summarized in Figure 2, which shows pooled data (aggregation rate at 25°C and 35°C) as a function of measured pH (excluding the pH 7 formulation, for which aggregation behavior is atypical). Figure 2 shows an overall non-linear increase in the rate of aggregation with increasing measured pH.

[0227] [Table 6]

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

[0229] The percent change in fragmentation after 4 weeks of storage at 2-8°C is presented in Table 7. Fragmentation rate constants, along with the corresponding standard errors (SE), were determined for formulations at 25°C and 35°C (from three time points) by multiple linear regression using 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 between 2 and 8°C was generally low. However, the rate of fragmentation increased with storage temperature and pH (pH ≥ 6.0). A more pronounced change in %LMW was observed between 2 and 8°C with formulations at ~pH 7. At 25 and 35°C, the rate of fragmentation was also highest for formulation 8 at ~pH 7 (despite the lowest protein concentration at 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 trends are summarized in Figure 4, which shows pooled data as a function of measured pH (fragmentation rates at 35°C with standard errors). As observed with 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 formation of acidic species and pH / buffer / salt over time (cation exchange chromatography) The formation of acidic species was 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 % 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) (with the exception of the histidine formulation at ~pH 7). The same trend was observed for formulations stored at 25°C.

[0234] The % change in acidic species after 4 weeks of storage at 2-8°C is shown in Table 8. The rate constants for acidic species formation, along with the corresponding standard errors (SE), were determined for formulations at 25°C and 35°C (from three time points) by multiple linear regression using 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 also summarized in Table 8.

[0235] The data revealed that fragmentation occurring between 2 and 8°C was generally low. However, the rate of acidic species formation increased with storage temperature and pH (pH ≥ 6.0). The highest levels of acidic species were formed after 4 weeks at 2-8°C in formulations at ~pH 7. 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 buffers exhibiting greater storage stability compared to formulations at the same pH in acetate or citrate buffers.

[0237] The addition of NaCl (high ionic strength) showed more storage stability in terms of the rate of formation of acidic species than the formulation at the same pH without salt.

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

[0239] [Table 8]

[0240] Conclusions of Examples 1 to 10 The results showed that formulation of high-concentration (100 mg / mL) Fc multimeric molecule (CSL730) required specific conditions to prevent protein precipitation. A pH of ≥ 6.5 was not suitable for formulation of high-concentration (100 mg / mL) Fc multimeric molecule (CSL730) due to precipitation. High-protein concentration (100 mg / mL) Fc multimeric molecule (CSL730) formulations were more stable to aggregation, fragmentation, and acidic species formation at pHs below 6. Furthermore, high-protein concentration (100 mg / mL) Fc multimeric molecule (CSL730) formulations were more stable to high salt concentrations and acidic species formation in histidine buffers.

[0241] Part 2 - Stabilizer Screening Test An excipient screening study was conducted to examine the effects of stabilizer type (sugars / polyols and amino acids), stabilizer level, surfactant level, antioxidant type, and antioxidant level on liquid formulations containing Fc multimeric molecules (CSL730) at protein concentrations ranging from 10 to 100 mg / mL. A parallel short-term (3-month) study was conducted to examine storage stability as a function of protein concentration. Overall, CSL730 formulations with protein concentrations ranging from 10 to 100 mg / mL were prepared and protein stability was examined.

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

[0243] [Table 9]

[0244] Formulation Preparation: Formulations were 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 buffer of choice 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 surfactant of choice 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, spanning a target concentration range of 200-300 mM. Four formulations contained different levels (1-20 mM) of antioxidant (either methionine or reduced glutathione GSH). The study (and therefore the formulation differences) was designed to primarily modify the one-factor-at-a-time (OFAT) method. A summary of the formulations prepared for this study is shown in Table 10.

[0246] The following buffers 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 listed above. The pH of each buffer was measured after preparation. A 30% excess (mg protein) was applied to compensate for losses. The formulations were dialyzed against approximately 1 L of buffer for approximately 5 hours, followed by replacement with fresh buffer (approximately 1 L) and overnight dialysis. Dialysis was performed in 30 or 70 mL 10K MWCO cassettes at 2-8°C.

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

[0249] The following stock solutions were prepared and used as spiking solutions to aid in the preparation of the final formulation: 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 "CSL730 Diluent")

[0250] The drug substance (DS) consisted of 118 mg / mL recombinant Fc multimer CSL730, 20 mM histidine, 40 mM NaCl, 200 mM proline, and 0.02% w / v PS80 at pH 6. The DS was diluted by adding the appropriate volume of stock solution 1 or stock solution 2 to a protein concentration of 100 ± 5 mg / mL, followed by adjusting the pH 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 the DS to 100 ± 5 mg / mL with stock solution 4, while maintaining the pH at 5.8 ± 0.1.

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

[0252] Filling and finishing 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 labeled 2 ml glass vials. The vials were then stoppered and crimped. A total of 17 liquid formulations and 13 placebos were prepared.

[0253] After the fill / finishing step, vials of the filled liquid formulation were placed in stability chambers at 2-8°C, 25°C, or 40°C. For the "T0" / T=0 analysis, samples were analyzed immediately after the fill / finishing step and then at predetermined subsequent time points up to 6 months. To compare the degradation kinetics between different formulations, rate constants (with standard errors) for the different pathways of degradation were determined using multiple linear regression using JMP and Excel software. The multiple linear regression was calculated using a general linear model (GLM), and the 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: The formulations were analyzed at different time points for oxidation using stability-indicating methods, mainly: size exclusion chromatography (SEC), cation exchange chromatography (CEX), capillary gel electrophoresis (CGE) (Caliper) and reverse phase HPLC (RP-HPLC). Additionally, the pH, appearance (color, turbidity and visible particles) and subvisible particles (SVPs) of the formulations were monitored at different time points.

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

[0257] [Table 11]

[0258] [Table 12]

[0259] The analysis method is explained 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] Osmolality: The osmolality of the formulations was measured at the starting time point (T0) by freezing point depression using a Gonotec Osmomat 3000 Osmometer. The sample volume was 300 μL. Measurements were performed in triplicate per formulation, and the mean value of the measurements was calculated.

[0261] Subvisible particle count test: A limited analysis of subvisible particles using a FlowCam Biologics device (Dynamic / Flow Imaging Particle Analysis - DIPA - technology) was performed on the formulations of interest only after a storage time of 3 months, at 2-8°C and 25°C. A minimum sample volume of 0.5 mL was used. Measurements were performed in triplicate per formulation and the mean of the measurements was calculated.

[0262] Analysis of oxidation by reversed-phase high-performance liquid chromatography (RP-HPLC): The RP-HPLC method was used to determine the total amount of oxidized species as a percentage of the total area, and the relative amounts of both short- and long-chain oxidation. Sample preparation involved diluting the sample to 10 g / L in an appropriate buffer, followed by the use of guanidine as a denaturing reagent and DL-dithiothreitol (DTT) for the reduction of disulfide bonds. This step generates two identical long chains and two identical short chains. 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 x 50 mm column. Separation was performed using a 0.35 ml / min gradient with a 3 μL injection volume. The column temperature was set to 65°C. Briefly, two buffers (0.1% trifluoroacetic acid in water and 0.08% TFA in acetonitrile) were exchanged over 20 min. Species were detected at 280 nm, identified against reference standards, and reported as relative area percentages relative to the integrated area.

[0263] Analysis of polysorbate 80 (PS80): The amount of PS80 at the starting time point (T0) in different formulations was quantified using RP-HPLC. Briefly, PS80 standards and samples were treated with ethanol, followed by 0.1 M KOH at 40°C, and then the samples were analyzed for oleic acid resulting from hydrolysis using a reversed-phase HPLC method. Samples were analyzed using a Dionex (Ultimate 3000) system (or equivalent) equipped with a Nova-Pak® C18 3.9 x 150 mm, 4 μm reversed-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 with 20% potassium dihydrogen phosphate buffer at pH 2.8. The column temperature was set at 40°C. Species were detected at 250 nm and quantified using a standard calibration curve generated with a PS80 standard solution. Data were reported as % (w / v) of PS80.

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

[0265] With the exception of the 10 mg / mL formulation, all other formulations exhibited a slightly opalescent, slightly brownish yellow (BY) (70 mg / mL formulation) to BY (100 mg / mL formulation) color due to the high protein concentration. 0 to 2 visible particles were observed randomly in some formulations and placebo. Due to the protein findings, workup revealed that the visible particles were exogenous in nature (probably introduced during the filling / finishing process). The high protein concentration formulations did not gel.

[0266] Formulations 1-18 (see Table 10) were visually inspected for up to 6 months at different temperatures. The color of most formulations remained unchanged from TO 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 with reduced GSH were slightly more BY color compared to formulations with the same protein concentration. Clarity remained unchanged (clear for the 10 mg / ml formulation, slightly opalescent for all others). Particle counts remained unchanged (0-2) from TO regardless of temperature and time. 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 intensified with increasing storage time, storage temperature, and GSH concentration. The placebos of the other formulations remained clear and colorless regardless of temperature and time. Overall, particle counts remained unchanged from TO.

[0268] Example 2 Osmolality, pH, protein concentration and polysorbate 80 measurements at T0 and over time Osmolality, pH, protein concentration, and polysorbate 80 (PS80) concentration measurements for all formulations 1-18 (see Table 10) were performed at the starting time point (T0). The results are summarized in Table 13.

[0269] The results in Table 13 showed that protein concentrations were within 100 ± 5 mg / mL for the high-concentration formulations. pH measurements showed that the measured pH values ​​were within ± 0.1 pH units for all formulations compared to the target pH values. Osmolality measurements were generally within 300-390 mOsm / kg, except for the arginine-containing formulations (Formulations 5 and 6), which had osmolality values ​​exceeding 400 mOsm / kg. PS80 concentrations for all formulations were within a maximum 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 shifts in pH (within ±0.1 units maximum) 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 average fluctuations in protein concentration within the measurement period were 1.0 mg / mL from 2 to 8°C, 1.7 mg / mL at 25°C, and 1.6 mg / mL at 40°C. This is likely a result of the relative assay variability of the high-throughput technology used.

[0273] [Table 13]

[0274] [Table 14]

[0275] [Table 15]

[0276] Example 3 Limited analysis of subvisible particles at 6 months Low subvisible particle counts were measured for most formulations tested after 3 months of storage at both 5°C and 25°C for all particle size ranges tested (see Figure 7).

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

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

[0279] The initial (T0) % HMW for all formulations is shown in Table 17. Aggregation rate constants, along with corresponding standard errors (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 methods used to prepare different formulations. For example, some dialysates had to be concentrated after dialysis, 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 increasing protein concentrations (F2, F10, F11). Note that these formulations were formulated by simple dilution using the same buffer from the same stock dialysate solution.

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

[0282] As expected, the results in Table 16 show an increased rate of aggregation with storage temperature. For every 10°C increase in temperature, up to 40°C, a more pronounced change was observed, with an average 1.75-fold increase in aggregation rate, indicating a possible different mechanism of molecular aggregation at higher storage temperatures compared to lower storage conditions.

[0283] To evaluate the effect of stabilizer type, the storage stability of F1 to F7 was compared. The stabilizers used in the formulations were sugars / polyols, amino acids, or a mixture of amino acids and sugars. The total stabilizer level in these formulations was fixed at 250 mM. The results in Table 16 show that the Fc multimeric 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 slightly lower aggregation rates at 2-8°C and 25°C and significantly lower aggregation rates at 40°C. The sorbitol-based formulations showed superior stability to the amino acid-based formulations at 40°C (regardless of the presence or absence of sucrose with amino acids), but showed comparable stability at lower temperatures for up to 6 months. Similarly, the proline-based formulations showed superior stability to the arginine-based formulations at 40°C (regardless of the presence or absence of sucrose), but showed comparable stability at lower temperatures for up to 6 months.

[0284] To evaluate the effect of protein concentration at fixed stabilizer levels, the storage stability of F2, F10, and F11 was compared using sucrose as a glycoform stabilizer. Protein concentrations ranged from 10 to 100 mg / mL. As expected, the results in Table 16 show that the Fc multimeric molecule (CSL730) exhibited excellent stability at very low concentrations in F11 (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 protein concentration (results not shown). Note that at fixed stabilizer levels, the molar ratio of stabilizer to protein increased as protein concentration decreased (Table 10), a factor that may contribute to the significant improvement in storage stability.

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

[0286] To evaluate the effect of antioxidant type and level at fixed stabilizer levels, 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 with methionine as an antioxidant, the Fc multimeric molecule (CSL730) showed incremental (small) improvements in storage stability (physical) with increasing methionine levels at 40°C. However, there was no significant difference between the physical stability of formulations with and without methionine. The formulation containing reduced GSH showed significantly greater instability at 40°C compared to the formulation without antioxidant (F2) and the formulations containing methionine as an antioxidant (F12, F13). Although slight differences in storage stability were observed between GSH-containing formulations compared to F2, F12, and F13 at 25°C, the results were similar (lower stability with reduced GSH formulations). Nevertheless, reduced GSH formulations (F14, F15) were excluded after 3 months of storage stability because stability data by other methods showed significant instability for F14 and F15, warranting their exclusion.

[0287] To evaluate the effect of surfactant (PS80) level on the immobilized 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 differences in the storage stability (aggregation) of both formulations were observed at 2-8°C and 25°C for up to 6 months. However, the Fc multimeric molecule (CSL730) was more stable at the lower PS80 concentration (F2) at 40°C.

[0288] [Table 16]

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

[0290] The initial (TO) %LMW total species of all formulations was measured. F15 had the highest TO level of fragments, an observation confirmed using SEC (data not shown). The initial (total) %LMW species measured for all formulations, except F15, were relatively low, ranging between 2.1 and 2.5%.

[0291] To select formulations, except for F10 and F11, which are relatively high-throughput methods, the fragmentation behavior of the formulations was monitored over time at different temperatures using the NR-cGE "Caliper" method. Fragmentation rate constants, along with the corresponding standard errors (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 discontinued after 3 months of storage at 40°C because fragmentation in all formulations reached a level sufficient to distinguish between formulation performance in terms of fragmentation stability (best to worst performance). Analysis of F14 and F15 was also discontinued after 3 months at 2-8°C and 25°C because they exhibited weak enough stability to warrant their exclusion by this and other methods (aggregation, oxidation). In general, as observed by SEC analysis, the % major species was observed to decrease over time, with a concomitant increase in aggregates (% HMW) and fragments (% LMW). However, aggregation is not reported by this method because it is reported by SEC, a more robust method for soluble aggregates.

[0293] Small variations in fragmentation (within assay error) occurred in all formulations tested at 2-8°C over time, as evidenced by very small changes in %LMW after 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 stabilizer type, the storage stability of F1 to F7 was compared. The results in Table 17 show that the Fc multimeric molecule (CSL730) exhibited a more pronounced fragmentation potential in the presence of arginine (regardless of the presence or absence of sucrose: F5 and F6) at both 25°C and 40°C. Fragmentation was comparable in all other formulations at 25°C. At 40°C, less fragmentation was observed in the sucrose and sorbitol formulations (F2 and F4, respectively) compared to the 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 level at a fixed protein concentration (100 mg / mL), the storage stability of F2, F8, and F9 was compared using sucrose as a glycoform stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 17 showed no significant differences in fragmentation stability at 25°C as a function of sucrose level. Surprisingly, the formulation with 250 mM sucrose (F2) exhibited greater fragmentation stability at 40°C than the corresponding formulations with 225 mM and 300 mM sucrose. With proline as a stabilizer, the Fc multimeric molecule (CSL730) (F17) in 200 mM proline exhibited slightly better stability with respect to fragmentation at 25°C and 40°C compared to the formulation with 250 mM proline.

[0297] To evaluate the effect of antioxidant type and level on the fixed 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 multimeric molecule (CSL730) at 40°C in the presence versus absence of methionine as an antioxidant. The stability with respect to fragmentation was slightly better in the absence of methionine at 25°C for reasons the inventors cannot explain. More significant instability with respect to fragmentation was observed with formulations containing reduced GSH only after 3 months of storage at both 25°C and 40°C. Therefore, the reduced GSH formulations (F14, F15) were excluded after 3 months of storage because the stability data indicated sufficient instability of F14 and F15 to warrant their exclusion by this and other methods.

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

[0299] [Table 17]

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

[0301] Figure 8 compares the starting (T0) results for % acidic and basic species for all formulations. Most formulations had comparable % acidic species at T0. F15 (higher reduced GSH levels) and F17 (lower proline levels) had starting levels of % acidic species that were ~2.5-3.5% lower compared to the other formulations (see also Table 9). Similarly, most formulations had comparable % basic species at T0 (~4-5%). However, F15 had the highest starting level of % basic species (6.1%) compared to the other formulations.

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

[0303] Analysis of all formulations was discontinued after 2 months of storage at 40°C because the levels of acidic species in all formulations were sufficiently different and high to distinguish between formulation performance with respect to acidic species stability (best to worst performance). Analysis of F10, F11, F14, and F15 was also discontinued after 3 months at 2-8°C and 25°C. F14 and F15 demonstrated sufficiently weak stability to warrant their exclusion by this and other methods. In general, the % major species was observed to decrease over time with a concomitant increase in the % basic species and a greater increase in the % 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 (within assay error) in % acidic species were observed with other formulations at the same temperature. For formulations continued after the 3-month time point at 2-8°C, small variations in % acidic species were observed within the same formulation. Of 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 that the rate of formation of acidic species increased with storage temperature.

[0306] To evaluate the effect of the type of stabilizer, the storage stability of F1 to F7 was compared. The results in Table 18 show that the Fc multimeric 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 tendency of fragmentation and aggregation stability).

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

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

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

[0310] To evaluate the effect of surfactant (PS80) level on the fixed stabilizer level, the storage stability of F2 (0.02% w / v PS80) was compared with that of F18 (0.04% w / v PS80). Small differences were observed between the storage stability (acidic species formation) of both formulations at 25°C and 40°C.

[0311] [Table 18]

[0312] Example 6 Examination of oxidation behavior by reversed-phase high-performance liquid chromatography (RP-HPLC) at TO and over time All formulations (see Table 10) were analyzed by RP-HPLC to quantify the total % oxidation at TO and the % oxidation in long-chain (LC) and short-chain (SC) fragments, as described above. Figure 9 shows a representative chromatogram of the oxidation profile of an Fc multimeric molecule (CSL730) in a sample formulation. Figure 9 shows the elution of SC-oxidized species followed by SC, then LC-oxidized species followed by LC. The chromatographic profile also contains SC and LC fragments, as well as SC and LC N-tertiary pyruvates. A placebo sample was also run by the RP-HPLC method, and no interference from placebo components was observed in the chromatogram due to the protein peak (data not shown). This was particularly important for the run of formulations with reduced GSH.

[0313] Figure 10 compares the initial (TO) results for the relative area (%) of %SC oxidized species, %LC oxidized species, and total oxidation for all formulations. Most formulations had comparable %oxidized species at TO. Most formulations had comparable initial levels of %SC oxidized species, %LC oxidized species, and total oxidation. Meanwhile, higher levels of oxidation were observed by all measurements for formulations with reduced GSH (F14 and F15) (see also Table 19). Increasing reduced GSH levels increased the %oxidized species (SC and LC) and total %oxidation at TO.

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

[0315] A few select formulations were analyzed for up to 6 months at 2-8°C and 25°C. Oxidation analysis of some formulations was discontinued after only 2 months at 25°C (based on their performance at 40°C). Analysis of all formulations was discontinued after 2 months of storage at 40°C because oxidation had reached a high enough level to allow identification and exclusion of the worst-performing formulations. In general, % unoxidized SC and % unoxidized LC were observed to decrease over time with a concomitant increase in % oxidized SC and % oxidized LC species. Overall, total % oxidation in the different formulations increased at rates that varied over time and 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, which showed a small increase in total % oxidation. However, the formulation containing arginine as the base stabilizer (F5) showed the greatest oxidation after 6 months of storage, while F14 and F15 were not included at 2-8°C.

[0317] As expected, the results in Table 19 show that the rate of oxidation increased with storage temperature. Notably, 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 systems tested, oxidation becomes less temperature dependent and more driven by GSH concentration, thus resulting in altered oxidation kinetics.

[0318] To evaluate the effect of stabilizer type, the storage stability of F1 to F7 was compared. The results in Table 19 show that the Fc multimeric molecule (CSL730) exhibited greater stability against oxidation in the presence of polyols / sugars at 25°C compared to amino acids. However, the outcome at 40°C was not consistent with that at 25°C. Therefore, no trends could be concluded from the results. Given the assay variability in the levels of oxidation measured, differences between formulations are likely small.

[0319] To evaluate the effect of protein concentration on the immobilized stabilizer level, the storage stability of F2, F10, and F11 was compared using sucrose as the glycoform stabilizer. The results in Table 19 show that the Fc multimeric molecule (CSL730) exhibited greater stability to oxidation as protein concentration increased 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-fold more stable than F11 at 40°C.

[0320] To evaluate the effect of stabilizer level at a fixed protein concentration (100 mg / mL), the storage stability of F2, F8, and F9 was compared using sucrose as a tower stabilizer. Stabilizer levels ranged from 200 to 300 mM. The results in Table 19 show that the Fc multimeric molecule (CSL730) showed little difference in oxidative stability between the three formulations with different sucrose levels at both 25°C and 40°C. The same outcome held true 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 antioxidant type and level at fixed stabilizer levels, the storage stability of F2, F12, F13, F14, and F15 was compared. The results in Table 19 show a small but systematic improvement in oxidative stability with increasing levels of methionine at 25°C compared to its absence (F12 and F13). A systematic but more significant improvement in oxidative 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 exhibited 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 2 months of storage at both 25°C and 40°C. At both temperatures, just 1 mM GSH resulted in 4.7- to 9.3-fold more oxidation than its equivalent formulation with methionine at both 25°C and 40°C. Therefore (as with other methods discussed previously), reduced GSH formulations (F14, F15) were excluded based solely on their oxidative storage stability of 2 months.

[0322] To evaluate the effect of surfactant (PS80) level on the fixed stabilizer level, the storage stability of F2 (0.02% w / v PS80) was compared with that of F18 (0.04% w / v PS80). No measurable difference was observed between the storage stability (oxidation) of both formulations at 40°C.

[0323] [Table 19]

[0324] Conclusion of Part 2 A stable liquid DP formulation of high concentration (≥100 mg / mL) CSL730 is suitable Stable against aggregation, acidic speciation, fragmentation, and oxidation Factors that contribute to the stabilization of Fc multimer molecules, especially at high protein concentrations ·Low pH (5~5.8) Use of disaccharides / polyols, and to a lesser extent amino acids, as stabilizers The optimal molar ratio for protein stability is a protein to sucrose ratio of 1:420. Stability was also achieved with lower protein to sucrose ratios. The strength of the formulation increased above that ratio. Antioxidants are needed to prevent yellowing and / or side-chain oxidation reactions that may occur with other excipients during storage, or to protect against certain manufacturing conditions (e.g., exposure to light sources, exposure to evaporated hydrogen peroxide residues from sterilization in isolator filling machines), or unusual handling conditions during clinical administration. High PS80 levels are not required for storage stability, but are required to ensure stability during manufacturing and clinical handling / administration.

[0325] Part 3: Stabilizer screening tests at protein concentrations of 120-160 mg / ml A follow-up study to the excipient screening study was conducted to examine the effect of stabilizer type (the stabilizers of interest based on Part II were sucrose, trehalose, and proline) on liquid formulations containing an Fc multimeric molecule (CSL730) at high protein concentrations in the presence of 20 mM histidine, 0.02% w / v polysorbate 80, and 10 mM methionine. While protein concentrations were varied, the protein-to-stabilizer molar ratio 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 protein stability was examined. The formulations prepared for this study are summarized in Table 20.

[0326] [Table 20]

[0327] Methods used to analyze the stability of the formulations The formulations were analyzed at different time points for oxidation using stability indicating methods, mainly: size exclusion chromatography (SEC), cation exchange chromatography (CEX), capillary gel electrophoresis (CGE) (Caliper) and reverse phase HPLC (RP-HPLC). Additionally, the pH, appearance (color, turbidity and visible particles) and subvisible particles (SVPs) of the formulations were monitored at different time points.

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

[0329] [Table 21]

[0330] [Table 22]

[0331] The analytical methods were performed as described in Parts 1 and 2 above.

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

[0333] Due to the high protein concentration, all formulations exhibit a slightly opalescent brownish-yellow (BY) color. 0-1 visible particles were observed randomly in some formulations and placebo. Due to the protein findings, workup revealed that the visible particles were exogenous in nature (probably introduced during the filling / finishing process). 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 TO at all storage temperatures (BY). The transparency remained unchanged (slightly opalescent). The particle count remained unchanged from TO (0-1) regardless of temperature and time. The formulations did not gel during storage at any temperature.

[0335] Example 2 Osmolality, pH, protein concentration and polysorbate 80 measurements at T0 and over time Osmolality, pH, protein concentration, and polysorbate 80 (PS80) concentration measurements for all formulations 1-6 (see Table 20) were performed at the starting time point (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 for most formulations compared to the target pH values. A slightly higher deviation in pH was observed for F6 (within 0.17 units). Osmolality was typically within 382–414 mOsm / kg for the 120 mg / mL formulations, while osmolality was typically higher for the 160 mg / mL formulations (529–551 mOsm / kg). PS80 concentrations for all formulations were within a maximum 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 shifts in pH (within ±0.1 units maximum) at all temperatures for all formulations within the measured time frame. Also within experimental error, the results showed small shifts in protein concentration over time.

[0338] [Table 23]

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

[0340] The initial (T0) % HMW for all formulations is shown in Table 24. Aggregation rate constants, along with corresponding standard errors (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 and low (within 0.8-1% HMW) despite the high protein concentration.

[0342] All formulations were analyzed for up to 12 months of storage at 2-8°C, 25°C, and up to 6 months of storage at 35°C. As expected, the results in Table 24 show that the rate of aggregation increased with storage temperature. Within the stability testing temperature range, the rate of aggregation increased by up to 2-fold for every 10°C increase in temperature.

[0343] The stability of the Fc multimeric molecule (CSL730) at a high protein concentration (160 mg / ml) was on average 1.3-fold less stable at 2-8°C, 25°C, and 35°C in contrast to the same formulation at a low protein concentration (120 mg / ml).

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

[0345] [Table 24]

[0346] [Table 25]

[0347] All F1 through 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 Examination of fragmentation behavior (low molecular weight species) by non-reducing capillary gel electrophoresis (NR-cGE) at TO and over time All formulations (see Table 20) were analyzed by non-reducing cGE using Caliper (NR Caliper) for quantitation of low molecular weight (LMW) species (i.e., fragmentation) at TO, as described above.

[0349] The initial (T0) %LMW species for all formulations are shown in Table 25. As shown in Table 25, the %LMW at T0 for all formulations was comparable. The same outcome was observed for the LMW measured by the SEC method at T0 (%LMW measured for all formulations ranged from 2.6 to 2.7%).

[0350] The %LMW species of all formulations were monitored over time at different temperatures using the NR-cGE "Caliper" method, initially for 3 months and now continued for 12 months. Fragmentation rate constants, along with corresponding standard errors (SE), were 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 that the rate of fragmentation increases with storage temperature. Small variations in fragmentation (within assay error) occurred for all formulations tested at 2-8°C over time, as evidenced by small changes in %LMW over the course of up to 12 months. However, with each 10°C increase in storage temperature (from 25°C to 35°C), the rate of fragmentation increased an average of 3.6-4 fold, regardless of protein concentration. Similar results were observed with the rate of fragmentation measured by SEC (results not shown). With each 10°C increase in storage temperature (from 25°C to 35°C), the rate of fragmentation increased an average of 3.5 fold by SEC.

[0352] At the same storage temperature, no difference was observed between the stability of the Fc multimeric molecule (CSL730) at a high protein concentration (160 mg / ml) in contrast to the same formulation at a low protein concentration (120 mg / ml). That is, there was no significant difference between the stability of the formulations at both 25°C and 35°C as a function of protein concentration. The same outcome was observed with LMW measured by SEC (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 multimeric molecule (CSL730) against fragmentation at 2-8°C, 25°C, and 35°C.

[0354] [Table 26]

[0355] [Table 27]

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

[0357] The initial (T0) % acidic species for all formulations is shown in Table 26. As shown in Table 26, the % acidic species at T0 for all formulations was comparable. Similarly, the % basic species at T0 for all formulations was also comparable (3.5-3.7%).

[0358] The % acidic species of all formulations were monitored over time (up to 12 months) at different temperatures using the CEX method. The rate constants for the formation of acidic species, along with the corresponding standard errors (SE), were 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 that the rate of formation of acidic and basic species increases with storage temperature. No change in acidic species formation was observed for any of the formulations tested 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 3.1-3.4 fold.

[0360] The results in Table 26 show that the stability of the Fc multimeric molecule (CSL730) at a low protein concentration (120 mg / ml) was slightly better than 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 multimeric molecule (CSL730) with respect to the formation of acidic species in the presence of sucrose, trehalose, or proline at a fixed molar ratio of approximately 1:420 at 25° C. The results at 35° C. are inconclusive due to the small number of samples analyzed at that temperature.

[0362] [Table 28]

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

Claims

1. 1. An Fc multimer composition comprising: a) Fc multimers at a concentration between 60 mg / ml and 180 mg / ml; b) a pH value between 4.8 and 6.0; c) a buffer solution, wherein the buffer solution is an acetate buffer solution, a histidine buffer solution, or a citrate buffer solution; There are, and d) a stabilizer at a concentration between 200 mM and 450 mM, wherein the stabilizer is a polyol and / or an amino acid, Including, An Fc multimer is composed of three Fc moieties, each of which is a molecule assembled from two Fc polypeptides capable of binding to an Fc receptor, and each Fc polypeptide is a polypeptide chain consisting of a portion of the heavy chain constant domain of an antibody. An Fc polypeptide is not any portion of an immunoglobulin that can act as an antigen recognition region, nor is it any portion of an immunoglobulin that can act as an antigen recognition region. does not contain any complementarity determining regions (CDRs); and The Fc multimer comprises two polypeptides having the amino acid sequence of SEQ ID NO: 23 and SEQ ID NO: The Fc multimer composition comprising two polypeptides having the amino acid sequences of SEQ ID NO:

24.

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, particularly 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 stabilizer 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, particularly methionine.

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

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

10. The Fc multimer according to 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. composition.

11. The Fc multimer composition according to any one of claims 1 to 10, for use in the treatment of an autoimmune disease or an inflammatory disease.

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