DAS181 variant composition

Abstract

The present application provides compositions comprising at least four DAS181 subspecies that can be separated by CEX-HPLC, methods of making DAS181 compositions available for human medical use comprising subjecting the compositions to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by CEX-HPLC, formulations of DAS181 or multiple subspecies compositions of DAS181, and methods of treating disease comprising administering any of the DAS181 compositions described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 263,441, filed November 2, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing The contents of the electronic sequence listing (208712000440seqlist.xml, size: 5,657 bytes, created on: October 20, 2022) are incorporated herein by reference in their entirety.

[0003] The present application relates to compositions comprising subspecies of DAS181 and methods of use thereof. [Background technology]

[0004] DAS181 is a recombinant sialidase protein that cleaves sialic acid, e.g., sialic acid located on the surface of endothelial cells in the human airway wall. Because many different viruses use sialic acid as a receptor to infect the epithelial cells, treatment with DAS181 can block viral entry and prevent viral infection and spread. DAS181 has demonstrated antiviral activity against multiple sialic acid-dependent viruses, making it uniquely suitable as a broad-spectrum treatment against many different respiratory viral infections. Various compositions comprising DAS181 are described in US 8,084,036, US 7,807,174, US 9,764,007, US 7,645,448, US 8,152,710, US 8,722,869, US 10,525,109, US 2015 / 0132274, US 9,700,602, and US 10,351,828, the contents of each of which are incorporated herein by reference in their entirety. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,084,036 [Patent Document 2] U.S. Pat. No. 7,807,174 [Patent Document 3] U.S. Pat. No. 9,764,007 [Patent Document 4] U.S. Pat. No. 7,645,448 [Patent Document 5] U.S. Patent No. 8,152,710 [Patent Document 6] U.S. Pat. No. 8,722,869 [Patent Document 7] U.S. Pat. No. 10,525,109 [Patent Document 8] US Patent Application Publication No. 2015 / 0132274 [Patent Document 9] U.S. Patent No. 9,700,602 [Patent Document 10] U.S. Pat. No. 10,351,828 Summary of the Invention [Means for solving the problem]

[0006] The present application provides compositions comprising variants of DAS181, and methods of publishing compositions comprising the DAS181 variants.

[0007] In some embodiments, provided herein are compositions comprising at least four subspecies of DAS181 that can be separated by cation exchange high performance liquid chromatography (CEX-HPLC), the compositions including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid position relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via the C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-397 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein.

[0008] In some embodiments, provided herein are compositions comprising at least four subspecies of DAS181, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-397 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein.

[0009] In some embodiments, the deaminated N residue contained in the second subspecies is N403.

[0010] In some embodiments according to any of the preceding compositions, the fourth subspecies comprises a methionine oxidized form of the first fusion protein.

[0011] In some embodiments according to any of the preceding compositions, the composition further comprises a fifth subspecies comprising a dehydrated form of the first fusion protein.

[0012] In some embodiments of any of the preceding compositions, the protein included in the fourth sub-species has at least about 40% of the sialidase activity compared to that of the first fusion protein.

[0013] In some embodiments of any of the preceding compositions, the protein included in the third sub-species has at least about 80% of the sialidase activity compared to that of the first fusion protein.

[0014] In some embodiments according to any of the preceding compositions, the third subspecies further comprises an oxidized or deaminated form of the second fusion protein.

[0015] In some embodiments of any of the preceding compositions, the protein included in the first subspecies has at least about 94% of the sialidase activity compared to that of the first fusion protein.

[0016] In some embodiments of any of the preceding compositions, the first subspecies further comprises a third fusion protein that lacks an N-terminal M residue compared to the first fusion protein.

[0017] In some embodiments of any of the preceding compositions, the ratio of the first fusion protein to the third fusion protein in the first subspecies is about 2:1.

[0018] In some embodiments of any of the preceding compositions, the protein included in the second subspecies has at least about 80% of the sialidase activity compared to that of the first fusion protein.

[0019] In some embodiments of any of the preceding compositions, the sialidase domain includes amino acids 1-394 of SEQ ID NO:1.

[0020] In some embodiments of any of the preceding compositions, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0021] In some embodiments of any of the preceding compositions, the second fusion protein comprises amino acids 1-406 of SEQ ID NO:1.

[0022] In some embodiments of any of the preceding compositions, the weight percentage of the first subspecies of total protein in the composition is about 50% to about 90%.

[0023] In some embodiments of any of the preceding compositions, the percentage of the second subspecies of total protein in the composition is about 10% to about 30%.

[0024] In some embodiments of any of the preceding compositions, the weight percentage of the third subspecies of the total protein in the composition is about 1% to about 15%.

[0025] In some embodiments of any of the preceding compositions, the weight percentage of the fourth subspecies of the total protein in the composition is about 0.1% to about 4%.

[0026] In some embodiments of any of the preceding compositions, the weight percentage of the fifth subspecies of the total protein in the composition is about 1% to about 10%.

[0027] In some embodiments of any of the preceding compositions, when the composition is subjected to cation exchange chromatography using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid (CEX-HPLC) and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the first subspecies elutes at about 80-85 mM CaCl.

[0028] In some embodiments of any of the preceding compositions, when the composition is subjected to cation exchange chromatography using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid (CEX-HPLC) and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the second subspecies elutes at about 70-75 mM CaCl.

[0029] In some embodiments of any of the preceding compositions, when the composition is subjected to cation exchange chromatography using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid (CEX-HPLC) and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the third subspecies elutes at about 57-58 mM CaCl.

[0030] In some embodiments of any of the preceding compositions, when the composition is subjected to cation exchange chromatography using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid (CEX-HPLC) and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the fourth subspecies elutes at about 180-200 mM CaCl.

[0031] In some embodiments of any of the preceding compositions, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the fifth subspecies elutes at about 95-105 mM CaCl.

[0032] In some embodiments of any of the preceding compositions, the second fusion protein is capable of binding to a cell surface of an airway epithelium.

[0033] In some aspects, provided herein is a pharmaceutical composition comprising the composition of any of the preceding embodiments.

[0034] In some aspects, provided herein is a method of releasing a composition of any one of the preceding embodiments for human medical use, the method comprising subjecting the composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC.

[0035] In some embodiments of the disclosed methods, a weight percentage of the first subspecies of the total protein in the composition of about 50% to about 90% indicates that the composition is suitable for medical use in humans.

[0036] In some embodiments according to any of the preceding published methods, a weight percentage of the second subspecies of the total protein in the composition of about 15% to about 30% indicates that the composition is suitable for human medical use.

[0037] In some embodiments according to any of the preceding published methods, a weight percentage of the third subspecies of the total protein in the composition of about 1% to about 15% indicates that the composition is suitable for human medical use.

[0038] In some embodiments according to any of the preceding published methods, a weight percentage of the fourth subspecies of the total protein in the composition of about 0.1% to about 4% indicates that the composition is suitable for human medical use.

[0039] In some embodiments according to any of the preceding published methods, a weight percentage of the fifth subspecies of the total protein in the composition of about 1% to about 10% indicates that the composition is suitable for human medical use.

[0040] In some embodiments according to any of the prior published methods, the CEX-HPLC comprises using non-porous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluting with a gradient containing up to 200 mM CaCl in acetate buffer.

[0041] In some aspects, provided herein are pharmaceutical compositions disclosed for human medical use by any of the preceding disclosed methods.

[0042] In some aspects, provided herein is a method of making a pharmaceutical composition comprising the composition of any one of the preceding embodiments, the method comprising: a) introducing a nucleic acid encoding a protein of SEQ ID NO:1 into a bacterial host cell; b) expressing in the bacterial host cell the protein encoded by the nucleic acid; c) purifying the protein by chromatography to obtain a purified protein composition; and d) evaluating the suitability of the purified protein composition for human medical use, wherein the evaluating comprises subjecting the purified protein composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC.

[0043] In some embodiments, the method further comprises formulating the purified protein composition to obtain a pharmaceutical composition.

[0044] In some embodiments according to any of the preceding methods of making, the step of formulating the purified protein composition occurs after the step of evaluating the suitability of the purified protein composition.

[0045] In some embodiments according to any of the preceding methods of making, the step of formulating the purified protein composition is performed prior to the step of evaluating the suitability of the purified protein composition.

[0046] In some aspects, provided herein is a pharmaceutical composition made by any of the preceding methods of making.

[0047] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition comprises at least about 70% trehalose by dry weight.

[0048] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition comprises at least about 0.2% MgSO4 by dry weight.

[0049] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition comprises: a) about 95-98% w / w trehalose, b) about 0.2-0.4% w / w MgSO4, c) about 0.4-0.6% w / w sodium acetate, and d) about 0.1-0.3% w / w acetic acid.

[0050] In some embodiments, provided herein is a pharmaceutical composition comprising: a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain; b) about 95-98% w / w trehalose; c) about 0.2-0.4% w / w MgSO4; d) about 0.4-0.6% w / w sodium acetate; and e) about 0.1-0.3% w / w acetic acid.

[0051] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition is formulated as a lyophilized formulation.

[0052] In some embodiments according to any of the preceding pharmaceutical compositions, the composition does not include histidine or CaCl2.

[0053] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition comprises about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid.

[0054] In some embodiments according to any of the preceding pharmaceutical compositions, the potency of the sialidase contained in the pharmaceutical composition is about 540-740 U / mg protein.

[0055] In some embodiments of any of the preceding pharmaceutical compositions, the pharmaceutical composition after reconstitution into a liquid formulation has an osmolality of about 270-330 mOsm / kg.

[0056] In some embodiments of any of the preceding pharmaceutical compositions, the pharmaceutical composition after reconstitution into a liquid formulation has a viscosity of about 1.27-1.39 cps.

[0057] In some embodiments according to any of the preceding pharmaceutical compositions, the pharmaceutical composition after reconstitution into a liquid formulation has a pH of about 4.5 to about 6.5.

[0058] In some embodiments according to any of the preceding pharmaceutical compositions, at least about 95% of the protein contained in the pharmaceutical composition is monomeric.

[0059] In some embodiments, provided herein is a liquid formulation reconstituted from any one of the preceding pharmaceutical compositions.

[0060] In some aspects, provided herein is a vial containing any one of the preceding pharmaceutical compositions. In some embodiments, the vial is for single use.

[0061] In some aspects, provided herein is a nebulizer comprising a liquid formulation, the liquid formulation being reconstituted from any one of the preceding pharmaceutical compositions.

[0062] In some embodiments, provided herein is a commercial batch comprising any one of the preceding pharmaceutical compositions.

[0063] In some aspects, provided herein are methods of treating a disease in an individual, the methods comprising administering to the individual an effective amount of any of the preceding pharmaceutical compositions or a liquid formulation reconstituted from the pharmaceutical composition. [Brief description of the drawings]

[0064] [Figure 1] 1 shows representative CEX-HPLC profiles of compositions comprising the first, second, third, fourth, and fifth DAS181 subspecies described herein.

[0065] [Diagram 2] The intact mass analysis of the first subspecies is shown.

[0066] [Diagram 3] An intact mass analysis of the second subspecies is shown.

[0067] [Figure 4] An intact mass analysis of the third subspecies is shown.

[0068] [Diagram 5] 1 shows an intact mass analysis of the fourth subspecies.

[0069] [Figure 6] Size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) analysis of the fourth subspecies is shown.

[0070] [Figure 7] Sodium dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE) analysis of DAS181 variants detected by Coomassie blue staining is shown compared to the reference standard DAS181.

[0071] [Figure 8A] 1 shows a comparison of cation exchange high performance liquid chromatography (CEX-HPLC) of a lyophilized microparticle formulation of a liquid reconstituted composition at time 0 and after 2 weeks of incubation at 25° C. and 60% relative humidity.

[0072] [Figure 8B] 1 shows CEX-HPLC of a lyophilized cake formulation of a liquid reconstituted composition at time 0 and after 2 weeks of incubation at 25° C. and 60% relative humidity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] DAS181 protein comprises a sialidase domain and a charged anchor domain. During the manufacture of a DAS181 composition, variants of the DAS181 protein may be produced, such as DAS181 charged variants, conformational variants, deaminated variants, deamidated variants, truncated variants, oxidized variants, dimers, and / or multimers. The presence or absence of these variants and the specific amounts of these variants contained in the composition are highly dependent on the manufacturing process and may significantly affect the stability and suitability for clinical use of the DAS181 composition. The present application provides novel DAS181 compositions produced by a large-scale manufacturing process that has been proven to be stable and suitable for clinical use. The novel DAS181 compositions comprise DAS181 and specific DAS181 variants, and optionally excipients that are particularly suitable for formulating the DAS181 composition. Also provided are methods for assessing the suitability of a DAS181 composition for clinical use based on the presence / absence and / or relative amount of a DAS181 variant.

[0074] Thus, in one aspect, provided herein is a DAS181 composition comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid positions being relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some embodiments, the DAS181 composition further comprises a fifth subspecies comprising a dehydrated form of the first fusion protein.

[0075] In some embodiments, provided herein is a method of exposing a DAS181 composition for human medical use, the method comprising subjecting the composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by CEX-HPLC. Also provided herein are pharmaceutical compositions comprising a mixture of DAS181 subspecies, and methods of treating an individual comprising administering the same.

[0076] In another aspect, provided herein are lyophilized formulations of sialidase fusion proteins (e.g., DAS181 or a mixture comprising DAS181 and a DAS181 variant subspecies) prepared as a cake that can be reconstituted and administered via nebulization. In some embodiments, the lyophilized formulations comprise, by dry weight: a) about 1.1-1.5% w / w DAS181 or DAS181 and a DAS181 variant subspecies, b) about 95-98% w / w trehalose, c) about 0.2-0.4% w / w MgSO4, d) about 0.4-0.6% w / w sodium acetate, and e) about 0.1-0.3% w / w acetic acid. In some embodiments, the formulation comprises, by dry weight, about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid. In another aspect, provided herein is a liquid formulation of a sialidase fusion protein (e.g., DAS181 or a mixture comprising DAS181 and a DAS181 variant subspecies) reconstituted from any of the lyophilized formulations disclosed herein. In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 or DAS181 and a DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 or DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 or DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0077] I. Definition Terms are used herein as commonly used in the art unless otherwise defined below.

[0078] As used herein, "DAS181 variant" or "subspecies of DAS181" refers to a DAS181 protein (as set forth in SEQ ID NO: 1) or a variant or derivative thereof having a characteristic elution profile (i.e., under the same peak) by chromatography (e.g., CEX-HPLC) and / or a characteristic set of peaks by intact mass spectrometry. Individual "DAS181 variants" may include full-length DAS181 protein as set forth in SEQ ID NO: 1, and / or charge variants, conformational variants, deaminated variants, deamidated variants, truncated variants, oxidized variants, dimers, and / or multimers of DAS181. Individual variants may include one or more forms or variants of the DAS181 molecule having the amino acid sequence of SEQ ID NO: 1.

[0079] As used herein, "DAS181 variant" refers to a portion of a composition of DAS181 that has a distinct elution profile by chromatography (e.g., CEX-HPLC) (i.e., under the same peak) and / or a characteristic set of peaks by intact mass spectrometry compared to a pure, full-length, and unmodified DAS181 protein, i.e., a monomeric fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, which fusion protein has the amino acid sequence of SEQ ID NO: 1. DAS181 variants include, but are not limited to, charged variants, conformational variants, deaminated variants, deamidated variants, truncated variants, oxidized variants, dimers, and / or multimers of DAS181.

[0080] As used herein, "treatment" or "treating" refers to an approach to obtain a result, including a beneficial or desired clinical result. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease, preventing or slowing the onset or recurrence of the disease, slowing or slowing the progression of the disease, improving the condition of the disease, providing remission (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving quality of life, and / or prolonging survival. Also encompassed by "treatment" is the alleviation of the pathological consequences of the disease. The methods of this application contemplate any one or more of these aspects of treatment.

[0081] The terms "individual," "subject," and "patient" are used interchangeably herein to describe a mammal, including a human. In some embodiments, the individual is a human. In some embodiments, the individual is afflicted with cancer. In some embodiments, the individual is in need of treatment.

[0082] As understood in the art, an "effective amount" refers to an amount of a composition sufficient to produce a desired therapeutic result (e.g., reducing the severity or duration of cancer, stabilizing the severity of cancer, or eliminating one or more symptoms of cancer). For therapeutic use, beneficial or desired results include, for example, reducing one or more symptoms caused by the disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological phenotypes exhibited during the progression of the disease, improving the quality of life of those suffering from the disease, reducing the dose of other pharmaceutical agents required to treat the disease, enhancing the effect of another pharmaceutical agent, slowing the progression of the disease, and / or prolonging the survival of the patient. In some embodiments, an effective amount of a therapeutic agent may prolong survival (including overall survival and progression-free survival), produce an objective response (including a complete or partial response), alleviate to some extent one or more signs or symptoms of the disease or condition, and / or improve the quality of life of the subject.

[0083] The term "wild type" as used herein is a term of art understood by those of skill in the art and means the normal form of a naturally occurring organism, strain, gene or characteristic, as distinguished from a mutant or variant form.

[0084] The terms "non-naturally occurring" and "engineered" are used interchangeably and refer to the influence of human hand. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide is at least substantially free from at least one other component with which it is naturally associated in nature and found in nature.

[0085] As used herein, "sialidase" refers to a naturally occurring or engineered sialidase capable of catalyzing the cleavage of terminal sialic acids from the carbohydrate of a glycoprotein or glycolipid. As used herein, "sialidase" can refer to a domain of a naturally occurring or non-naturally occurring sialidase capable of catalyzing the cleavage of terminal sialic acids from the carbohydrate of a glycoprotein or glycolipid. The term "sialidase" also contemplates a fusion protein that includes a naturally occurring or non-naturally occurring sialidase protein, or an enzymatically active fragment or domain thereof, and another polypeptide, fragment or domain thereof, such as an anchor domain or a transmembrane domain.

[0086] As used herein, the term "sialidase" encompasses sialidase catalytic domain proteins. A "sialidase catalytic domain protein" is a protein that contains the catalytic domain of a sialidase or an amino acid sequence that is substantially homologous to the catalytic domain of a sialidase, but does not include the entire amino acid sequence of a sialidase. The catalytic domain is obtained when the sialidase catalytic domain protein substantially retains the functional activity of the intact sialidase from which the catalytic domain is derived. A sialidase catalytic domain protein can contain an amino acid sequence that is not derived from a sialidase. A sialidase catalytic domain protein can contain an amino acid sequence that is derived from or substantially homologous to the amino acid sequence of one or more other known proteins, or it can contain one or more amino acids that are not derived from or substantially homologous to the amino acid sequence of another known protein.

[0087] As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into an mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide are sometimes collectively referred to as the "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0088] "Percent (%) amino acid sequence identity" with respect to the polypeptide and antibody sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared after aligning the sequences, with any conservative substitutions considered as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. For purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004, each of which is incorporated herein by reference in its entirety for all purposes).

[0089] The term "polypeptide" or "peptide" is used herein to encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including, but not limited to, glycoproteins and all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.).

[0090] The term "pharmaceutical composition" refers to a preparation in a form such that the biological activity of the active ingredient contained therein is effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0091] "Pharmaceutically acceptable carrier" refers to one or more ingredients other than the active ingredient contained in a pharmaceutical formulation, which are non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, cryoprotectants, isotonicity agents, preservatives, and combinations thereof. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicological and manufacturing testing, and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration or other state / federal governments, or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in mammals, and more specifically, in humans.

[0092] The term "package insert" is used to refer to instructions customarily included in the commercial packaging of a therapeutic product that contain information about the indications, uses, dosage, administration, concomitant therapy, contraindications and / or warnings concerning the use of such therapeutic product.

[0093] An "article of manufacture" is any product (e.g., package or container) or kit that contains at least one reagent, e.g., an agent for the treatment of a disease or condition (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the article of manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

[0094] It will be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0095] Reference herein to "about" a value or parameter includes (and describes) a variation on that value or parameter per se. For example, a reference to "about X" includes the description of "X."

[0096] As used herein, a reference to a value or parameter "is not" generally means and describes a value or parameter "other than." For example, the method is not used to treat disease type X means that the method is used to treat a disease type other than X.

[0097] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0098] As used in this specification and the appended claims, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise.

[0099] As used herein, the term "and / or," phrases such as "A and / or B" are intended to include both A and B, A or B, A (alone), and B (alone). Similarly, as used herein, the term "and / or," phrases such as "A, B, and / or C" are intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0100] II. Composition The present application provides compositions comprising multiple subspecies of DAS181, including the first, second, third, fourth, and optionally fifth subspecies described in Sections 1.A, 1.B, 1.C, 1.D, and 1.E below.

[0101] In some embodiments, the composition includes a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain comprising a sequence comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid positions being relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via the C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein. In some embodiments, the composition further includes a fifth subspecies comprising a dehydrated form of the first fusion protein. In some embodiments, the composition is formulated into a lyophilized cake formulation. In some embodiments, the composition is formulated into a lyophilized microparticle formulation. In some embodiments, the lyophilized formulation is reconstituted into a liquid. In some embodiments, the DAS181 composition is a liquid composition (e.g., a reconstituted lyophilized composition). In some embodiments, the liquid composition further comprises 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 or DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 or DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0102] In some embodiments, when the compositions provided herein are subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in acetate buffer, the first subspecies elutes at about 80-87 mM CaCl2, the second subspecies elutes at about 70-75 mM CaCl2, the third subspecies elutes at about 56-59 mM CaCl2, and the fourth subspecies elutes at about 180-200 mM CaCl2. In some embodiments, the compositions further comprise a fifth subspecies, and when the compositions provided herein are subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in acetate buffer, the fifth subspecies elutes at about 90-110 mM CaCl2.

[0103] In some embodiments, the CEX-HPLC is performed using the following conditions: runtime 78 minutes, flow rate: 1 mL / min. In some embodiments, the CEX-HPLC is a first buffer (CEX-A) to a second buffer (CEX-B). In some embodiments, CEX-A is 19.5 mM sodium acetate and CEX-B is 19.5 mM sodium acetate and 200 mM CaCl2. In some embodiments, the CEX-HPLC is performed using the injection percentages of CEX-A and CEX-B shown in Table 1 below at a flow rate of 1 mL / min. [Table 1] In some embodiments, when the compositions provided herein are subjected to CEX-HPLC using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid and eluted according to the conditions set forth in Table 1, the first subspecies elutes at 40-45 minutes, 42-45 minutes, or about 44 minutes, the second subspecies elutes at 32-35 minutes, 33-35 minutes, or about 34 minutes, the third subspecies elutes at 12-16 minutes, 13-15 minutes, or about 14 minutes, and the fourth subspecies elutes at 58-62 minutes, 59-60 minutes, or about 59.5 minutes. In some embodiments, the compositions comprise a fifth subspecies, and the fifth subspecies elutes at 52-55 minutes, or 53-55 minutes, or about 52-55 minutes, or 53-55 minutes.

[0104] In some embodiments, when the compositions provided herein are subjected to cation exchange chromatography (CEX-HPLC) using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in acetate buffer, the first subspecies elutes at about 84 mM CaCl2, the second subspecies elutes at about 72 mM CaCl2, the third subspecies elutes at about 57.5 mM CaCl2, and the fourth subspecies elutes at about 200 mM CaCl2. In some embodiments, when the compositions provided herein are subjected to cation exchange chromatography (CEX-HPLC) using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in acetate buffer, the fifth subspecies elutes at about 100 mM CaCl2.

[0105] In some embodiments, the weight percentage of the first subspecies in the total protein of the composition is about 50% to about 90%, the weight percentage of the second subspecies in the total protein of the composition is about 5% to about 30%, the weight percentage of the third subspecies in the total protein of the composition is about 1% to about 15%, and the weight percentage of the fourth subspecies in the total protein of the composition is about 0.1% to about 15% or about 0.1% to about 4%. In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition is about 1% to about 15%. In some embodiments, the composition is suitable for human medical use.

[0106] In some embodiments, after storage at room temperature, the weight percentage of the first subspecies of total protein in the composition is at least about any one of 75%, 80%, 85%, 90% or more; the weight percentage of the second subspecies of total protein is at least about any one of 2%, 5%, 10%, 12% or more; the weight percentage of the third subspecies of total protein is at least about any one of 1%, 2%, 3%, 5%, 10% or more; and the weight percentage of the fourth subspecies of total protein is at least about any one of 1%, 2%, 3%, 5%, 10% or more. In some embodiments, after storage at room temperature, the weight percentage of the first subspecies of total protein in the composition is at most about any one of 90%, 85%, 80%, or 75%, the weight percentage of the second subspecies of total protein is at most about any one of 15%, 14%, 12%, or 10%, the weight percentage of the third subspecies of total protein is at most about any one of 30%, 20%, 15%, 14%, 12%, or 10%, and the weight percentage of the fourth subspecies of total protein is at most about any one of 30%, 20%, 15%, 14%, 12%, or 10%. In some embodiments, after storage at room temperature, the weight percentage of the first subspecies of the total protein in the composition is between about 80%-85%, 85%-90%, 75%-80%, or 75%-85%, the weight percentage of the second subspecies of the total protein is between about 5%-10%, 10%-15%, or 15%-20%, the weight percentage of the third subspecies of the total protein is between about 1%-5%, 5%-10%, 10%-15%, 15%-20%, or 20%-30%, and the weight percentage of the fourth subspecies of the total protein is between about 1%-5%, 5%-10%, 10%-15%, 15%-20%, or 20%-30%.In some embodiments, after storage at room temperature, the weight percentage of the first species of the total protein in the composition decreases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, after storage at room temperature, the weight percentage of the second, third, and / or fourth subspecies increases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, or 40% compared to before storage at room temperature. In some embodiments, the weight percentages are measured after storage at room temperature for at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the weight percentage is measured after storage at room temperature for at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours. In some embodiments, the weight percentage is measured after storage at room temperature for 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours-48 hours, 48 ​​hours-7 days, or 6 hours-7 days. In some embodiments, the composition is suitable for human medical use.

[0107] In some embodiments, the first subspecies comprises a variant of a first fusion protein, the first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1, the second subspecies comprises a deaminated form of the first fusion protein, and the fourth subspecies comprises a dimerized and / or methionine oxidized form of the first fusion protein. In some embodiments, the first fusion protein comprises a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1. In some embodiments, the first subspecies further comprises a third fusion protein lacking an N-terminal M residue compared to the first fusion protein. In some embodiments, the third fusion protein lacks an amino-terminal methionine compared to the first fusion protein.

[0108] The third subspecies includes a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain. "Truncated cationic domain" refers to a cationic domain that is truncated relative to the full-length cationic domain (amino acid residues 396-415) of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids in length. In some embodiments, the truncated cationic domain comprises any one of amino acid residues 396, 396-397, 396-398, 396-399, 396-400, 396-401, 396-402, 396-403, 396-404, or 396-495 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 12 amino acids in length and comprises amino acids 396-406 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 13 amino acids long and comprises amino acids 396-407 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 14 amino acids long and comprises amino acids 396-408 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 15 amino acids long and comprises amino acids 396-409 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 16 amino acids long and comprises amino acids 396-410 of SEQ ID NO:1.

[0109] In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1-394 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises a sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to amino acids 1-415 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0110] In some cases, the amino acid that differs from that of SEQ ID NO: 1 is a conservative substitution. A conservative substitution may be defined as an exchange within one of the following five groups: I. Small, aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln III. Polar, positively charged residues: His, Arg, Lys IV. Large, aliphatic nonpolar residues: Met, Leu, Ile, Val, Cys V. Large aromatic residues: Phe, Try, Trp

[0111] Within the aforementioned groups, the following substitutions are considered to be "highly conservative": Asp / Glu, His / Arg / Lys, Phe / Tyr / Trp, and Met / Leu / Ile / Val. Semi-conservative substitutions are defined as exchanges between two of the above groups (I) to (IV), which are limited to supergroup (A) including (I), (II), and (III) above, or supergroup (B) including (IV) and (V) above. Furthermore, when hydrophobic amino acids are specified in this application, they refer to the amino acids Ala, Gly, Pro, Met, Leu, Ile, Val, Cys, Phe, and Trp, and hydrophilic amino acids refer to Ser, Thr, Asp, Asn, Glu, Gln, His, Arg, Lys, and Tyr.

[0112] 1. DAS181 variant A. The first subspecies In some embodiments, the first subspecies comprises a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO: 1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO: 1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO: 1.

[0113] In some embodiments, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% by weight of the first subspecies is a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO: 1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1.

[0114] In some embodiments, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% by weight of the first subspecies is a first fusion protein, and the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0115] In some embodiments, about 50% to about 85%, about 60% to about 85%, about 65% to about 85%, or about 70% to about 85% by weight of the first subspecies is a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO: 1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO: 1.

[0116] In some embodiments, the first subspecies further comprises a third fusion protein lacking an N-terminal M residue compared to the first fusion protein. In some embodiments, the third fusion protein comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is about 1:1, about 1.2:1, about 1.3:1, about 1.4:1, about 1.5:1, about 1.6:1, about 1.7:1, about 1.8:1, about 1.9:1, about 2:1, about 2.1:1, about 2.2:1, about 2.3:1, about 2.4:1, about 2.5:1, about 2.6:1, about 2.7:1, about 2.8:1, about 2.9:1, or about 3:1. In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is about 2:1.

[0117] In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is at least about any of 1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, or 3:1. In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is at least about 1.5:1.

[0118] In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is at most about 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, or about 3:1. In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is at most about 2.5:1. In some embodiments, the ratio of the first fusion protein to the third fusion protein in the first subspecies is at most about 3:1.

[0119] In some embodiments, the ratio of the first fusion protein to the third fusion protein is about 1:1 to about 3:1, about 1.2:1 to about 2.9:1, about 1.5:1 to about 2.5:1, or about 1:1 to about 2:1.

[0120] In some embodiments, when a composition provided herein is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the first subspecies elutes at about 80-87 mM CaCl2. In some embodiments, when a composition provided herein is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the first subspecies elutes at about 80-85 mM CaCl2. In some embodiments, when a composition provided herein is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the first subspecies elutes at about 82-85 mM CaCl2. In some embodiments, when the compositions provided herein are subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl in acetate buffer, the first subspecies elutes at about 84 mM CaCl.

[0121] In some embodiments, the protein of the first subspecies has at least about any of 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, or 144% of the sialidase activity of the first fusion protein.

[0122] In some embodiments, the protein of the first subspecies has between 80% and 144%, 90% and 144%, 95% and 144%, 80% and 90%, 80% and 95%, 85% and 95%, 95% and 130%, 98% and 130%, or 99% and 130% of the activity of the protein of the first fusion protein. In some embodiments, the protein of the first subspecies has between 90% and 144% of the activity of the protein of the first fusion protein.

[0123] In some embodiments, the weight percentage of the first subspecies in the total protein contained in the composition is about 50% to about 90%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 50% to about 70%, or about 70% to about 95%. In some embodiments, the weight percentage of the first subspecies in the total protein contained in the composition is about 50% to about 90%. In some embodiments, the weight percentage of the first subspecies in the total protein contained in the composition is 70% to 85% or about 70% to 85%.

[0124] In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at least or at least about any one of 60%, 65%, 70%, 75%, 80%, or 85%. In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at most any one of 90%, 85%, or 80%.

[0125] In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the first subspecies in the total protein contained in the composition is about 80% to about 85%. In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the first subspecies in the total protein contained in the composition is 80% to 85%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the first subspecies in the total protein contained in the composition is about 80% to about 85%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the first subspecies in the total protein contained in the composition is 80% to 85%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the first subspecies in the total protein contained in the composition is about 80% to about 85%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the first subspecies in the total protein contained in the composition is 80% to 85%. In some embodiments, after storage at room temperature for about 3 days (e.g., 70-74 hours), the weight percentage of the first subspecies in the total protein contained in the composition is about 80% to about 85%. In some embodiments, after storage at room temperature for about 3 days (e.g., 70-74 hours), the weight percentage of the first subspecies in the total protein contained in the composition is 80% to 85%. In some embodiments, after storage at room temperature for about 7 days (e.g., 166-170 hours), the weight percentage of the first subspecies in the total protein contained in the composition is about 75% to about 80%. In some embodiments, after storage at room temperature for about 7 days (e.g., 166-170 hours), the weight percentage of the first subspecies in the total protein contained in the composition is about 78% to about 80%.

[0126] In some embodiments, after storage at room temperature, the weight percentage of the first subspecies in the total protein of the composition is at least about any one of 75%, 80%, 85%, 90%, or more. In some embodiments, after storage at room temperature, the weight percentage of the first subspecies in the total protein of the composition is at most about any one of 90%, 85%, 80%, or 75%. In some embodiments, after storage at room temperature, the weight percentage of the first subspecies in the total protein of the composition is at most about any one of 80%-85%, 85%-90%, 75%-80%, or 75%-85%. In some embodiments, after storage at room temperature, the weight percentage of the first species in the total protein of the composition is reduced by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, after storage at room temperature, the weight percentage of the first species of the total protein in the composition is reduced by about any one of 1-20%, 1-5%, 1-10%, 10-20%, or 5-15%. In some embodiments, the weight percentage is measured after storage at room temperature for at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the weight percentage is measured after storage at room temperature for at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours. In some embodiments, the weight percentage is measured after storage at room temperature for 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours to 48 hours, 48 ​​hours to 7 days, or 6 hours to 7 days.

[0127] In some embodiments, the first subspecies exhibits a first peak having a mass by intact mass analysis between 44647.42 Da and 44667.42 Da. In some embodiments, the first subspecies exhibits a first peak having a mass by intact mass analysis between 44652.42 Da and 44662.42 Da. In some embodiments, the first subspecies exhibits a first peak having a mass by intact mass analysis between 44653.42 Da and 44661.42 Da. In some embodiments, the first subspecies exhibits a first peak having a mass by intact mass analysis between 44654.42 Da and 44660.42 Da. In some embodiments, the first subspecies exhibits a first peak having a mass by intact mass analysis between 44654.92 Da and 44659.92 Da.

[0128] In some embodiments, the first subspecies exhibits a second peak having a mass by intact mass analysis between 44778.61 Da and 44798.61 Da. In some embodiments, the first subspecies exhibits a second peak having a mass by intact mass analysis between 44783.61 Da and 44793.61 Da. In some embodiments, the first subspecies exhibits a second peak having a mass by intact mass analysis between 44784.61 Da and 44792.61 Da. In some embodiments, the first subspecies exhibits a second peak having a mass by intact mass analysis between 44785.61 Da and 44791.61 Da. In some embodiments, the first subspecies exhibits a second peak having a mass by intact mass analysis between 44786.11 Da and 44791.11 Da.

[0129] In some embodiments, the first subspecies exhibits a third peak having a mass by intact mass analysis between 44825.42 Da and 44845.42 Da. In some embodiments, the first subspecies exhibits a third peak having a mass by intact mass analysis between 44830.42 Da and 44840.42 Da. In some embodiments, the first subspecies exhibits a third peak having a mass by intact mass analysis between 44831.42 Da and 44839.42 Da.

[0130] In some embodiments, the third peak represents a gluconate adduct of the first peak. In some embodiments, the third peak has a mass 176-180 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass 177-179 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass 178 Da greater than the mass of the first peak.

[0131] In some embodiments, the first subspecies exhibits a fourth peak having a mass by intact mass analysis between 44956.63 Da and 44976.63 Da. In some embodiments, the first subspecies exhibits a fourth peak having a mass by intact mass analysis between 44961.63 Da and 44971.63 Da. In some embodiments, the first subspecies exhibits a fourth peak having a mass by intact mass analysis between 44962.63 Da and 44970.63 Da.

[0132] In some embodiments, the first subspecies exhibits a first peak having an intact mass analysis mass between 44647.42 Da and 44667.42 Da, a second peak having an intact mass analysis mass between 44778.61 Da and 44798.61 Da, and a third peak having an intact mass analysis mass between 44825.42 Da and 44845.42 Da. In some embodiments, the first subspecies further exhibits a fourth peak having an intact mass analysis mass between 44956.63 Da and 44976.63 Da.

[0133] In some embodiments, the first subspecies exhibits a first peak having an intact mass analysis mass between 44652.42 Da and 44662.42 Da, a second peak having an intact mass analysis mass between 44783.61 Da and 44793.61 Da, and a third peak having an intact mass analysis mass between 44830.42 Da and 44840.42 Da. In some embodiments, the first subspecies further exhibits a fourth peak having an intact mass analysis mass between 44961.63 Da and 44971.63 Da.

[0134] In some embodiments, the first subspecies exhibits a first peak having an intact mass analysis mass between 44653.42 Da and 44661.42 Da, a second peak having an intact mass analysis mass between 44784.61 Da and 44792.61 Da, and a third peak having an intact mass analysis mass between 44831.42 Da and 44839.42 Da. In some embodiments, the first subspecies further exhibits a fourth peak having an intact mass analysis mass between 44962.63 Da and 44970.63 Da.

[0135] B. The second subspecies In some embodiments, the second subspecies comprises a deaminated form of the first fusion protein, the first fusion protein being a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0136] In some embodiments, the second subspecies comprises a deamidated and / or deamidated form of the first fusion protein, the first fusion protein being a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1. In some embodiments, deamidation is the primary degradation pathway of the first fusion protein comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, the second subspecies is predominantly deamidated and retains full potency.

[0137] In some embodiments, the protein in the second subspecies has at least or at least about 80%, 85%, 90%, 95%, or 100% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 80% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 85% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 86% of the sialidase activity of the first fusion protein.

[0138] In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is about 5% to about 30%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is any one of about 5% to about 10%, about 5% to about 20%, about 10% to about 20%, about 10% to about 15%, or about 5% to about 15%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is about 10% to about 30%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at least or at least any one of about 5%, 10%, 15%, 20%, or 25%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at most or at most about 30%, 25%, or 20%.

[0139] In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at least or at least about any one of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 13%, or 14%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at least or at least about 1%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at least or at least about 5%. In some embodiments, the weight percentage of the second subspecies in the total protein in the composition is at least or at least about 10%.

[0140] In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the second subspecies elutes at about 70-75 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the second subspecies elutes at about 71-74 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the second subspecies elutes at about 72 mM CaCl2.

[0141] In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the second subspecies in the total protein contained in the composition is about 5% to about 15% or about 10% to about 15%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the second subspecies in the total protein contained in the composition is about 5% to about 15% or about 10% to about 15%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the second subspecies in the total protein contained in the composition is about 5% to about 15% or about 10% to about 15%. In some embodiments, after storage at room temperature for about 3 days (e.g., 70 to 74 hours), the weight percentage of the second subspecies in the total protein contained in the composition is about 5% to about 15% or about 10% to about 15%. In some embodiments, after storage at room temperature for about 7 days (e.g., 166-170 hours), the weight percentage of the second subspecies of the total protein in the composition is about 5% to about 15% or about 10% to about 15%.

[0142] In some embodiments, after storage at room temperature, the weight percentage of the second subspecies in the total protein of the composition is at least about any one of 2, 5%, 10%, 12%, or more. In some embodiments, after storage at room temperature, the weight percentage of the second subspecies in the total protein of the composition is at most about any one of 15%, 14%, 12%, or 10%. In some embodiments, after storage at room temperature, the weight percentage of the second subspecies in the total protein of the composition is at most about any one of 5%-10%, 10%-about 15%, or 15%-20%. In some embodiments, after storage at room temperature, the weight percentage of the second subspecies in the total protein of the composition increases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, the weight percentage is measured after storage at room temperature for at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the weight percentage is measured after storage at room temperature for at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours. In some embodiments, the weight percentage is measured after storage at room temperature for 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours-48 hours, 48 ​​hours-7 days, or 6 hours-7 days.

[0143] In some embodiments, the second subspecies exhibits a first peak having a mass by intact mass analysis between 44647.42 Da and 44667.42 Da. In some embodiments, the second subspecies exhibits a first peak having a mass by intact mass analysis between 44652.42 Da and 44662.42 Da. In some embodiments, the second subspecies exhibits a first peak having a mass by intact mass analysis between 44653.42 Da and 44661.42 Da. In some embodiments, the second subspecies exhibits a peak having a mass by intact mass analysis between 44654.42 Da and 44660.42 Da. In some embodiments, the second subspecies exhibits a peak having a mass by intact mass analysis between 44654.92 Da and 44659.92 Da.

[0144] In some embodiments, the second subspecies exhibits a second peak having a mass by intact mass analysis between 44778.61 Da and 44798.61 Da. In some embodiments, the second subspecies exhibits a second peak having a mass by intact mass analysis between 44783.61 Da and 44793.61 Da. In some embodiments, the second subspecies exhibits a second peak having a mass by intact mass analysis between 44784.61 Da and 44792.61 Da. In some embodiments, the second subspecies exhibits a second peak having a mass by intact mass analysis between 44785.61 Da and 44791.61 Da. In some embodiments, the second subspecies exhibits a second peak having a mass by intact mass analysis between 44786.11 Da and 44791.11 Da.

[0145] In some embodiments, the second subspecies exhibits a third peak having a mass by intact mass analysis of 44825.42 Da to 44845.42 Da. In some embodiments, the second subspecies exhibits a third peak having a mass by intact mass analysis of 44830.42 Da to 44840.42 Da. In some embodiments, the second subspecies exhibits a third peak having a mass by intact mass analysis of 44831.42 Da to 44839.42 Da. In some embodiments, the third peak represents a gluconate adduct of the first peak. In some embodiments, the third peak has a mass 176-180 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass 177-179 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass 178 Da greater than the mass of the first peak.

[0146] In some embodiments, the second subspecies exhibits a fourth peak having a mass by intact mass analysis between 44956.63 Da and 44976.63 Da. In some embodiments, the second subspecies exhibits a fourth peak having a mass by intact mass analysis between 44961.63 Da and 44971.63 Da. In some embodiments, the second subspecies exhibits a fourth peak having a mass by intact mass analysis between 44962.63 Da and 44970.63 Da.

[0147] In some embodiments, the second subspecies exhibits a first peak having an intact mass analysis mass between 44647.42 Da and 44667.42 Da, a second peak having an intact mass analysis mass between 44778.61 Da and 44798.61 Da, and a third peak having an intact mass analysis mass between 44825.42 Da and 44845.42 Da. In some embodiments, the second subspecies further exhibits a fourth peak having an intact mass analysis mass between 44956.63 Da and 44976.63 Da.

[0148] In some embodiments, the second subspecies exhibits a first peak having an intact mass analysis mass between 44652.42 Da and 44662.42 Da, a second peak having an intact mass analysis mass between 44783.61 Da and 44793.61 Da, and a third peak having an intact mass analysis mass between 44830.42 Da and 44840.42 Da. In some embodiments, the second subspecies further exhibits a fourth peak having an intact mass analysis mass between 44961.63 Da and 44971.63 Da.

[0149] In some embodiments, the second subspecies exhibits a first peak having an intact mass analysis mass between 44653.42 Da and 44661.42 Da, a second peak having an intact mass analysis mass between 44784.61 Da and 44792.61 Da, and a third peak having an intact mass analysis mass between 44831.42 Da and 44839.42 Da. In some embodiments, the second subspecies further exhibits a fourth peak having an intact mass analysis mass between 44962.63 Da and 44970.63 Da.

[0150] C. The third subspecies The third subspecies includes a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain. "Truncated cationic domain" refers to a cationic domain that is truncated relative to the full-length cationic domain (amino acid residues 396-415) of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids in length. In some embodiments, the truncated cationic domain comprises any one of amino acid residues 396, 396-397, 396-398, 396-399, 396-400, 396-401, 396-402, 396-403, 396-404, or 396-495 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 12 amino acids in length and comprises amino acids 396-406 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 13 amino acids long and comprises amino acids 396-407 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 14 amino acids long and comprises amino acids 396-408 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 15 amino acids long and comprises amino acids 396-409 of SEQ ID NO:1. In some embodiments, the truncated cationic domain is about 16 amino acids long and comprises amino acids 396-410 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the second fusion protein comprises amino acids 1-406 of SEQ ID NO:1. In some embodiments, the third subspecies further comprises an oxidized or deaminated form of the second fusion protein.

[0151] In some embodiments, the second fusion protein comprising the truncated cationic domain is capable of binding to the cell surface of airway epithelium. In some embodiments, the second fusion protein has at least or at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% binding to the cell surface of airway epithelium compared to the binding of the first fusion protein to the cell surface of airway epithelium. In some embodiments, the second fusion protein has at least or at least about 85% binding to the cell surface of airway epithelium compared to the binding of the first fusion protein to the cell surface of airway epithelium.

[0152] In some embodiments, the second fusion protein has 80%-90%, 85%-90%, 80%-100%, 80%-110%, 100%-120%, 120%-150%, or 95%-100% binding to the airway epithelial cell surface compared to the binding of the first fusion protein to the airway epithelial cell surface.

[0153] In some embodiments, the protein in the third subspecies has at least or at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the third subspecies has at least or at least about 80% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the third subspecies has at least or at least about 85% of the sialidase activity of the first fusion protein.

[0154] In some embodiments, the second fusion protein has 80%-90%, 85%-90%, 80%-100%, 80%-110%, 100%-120%, 120%-150%, or 95%-100% of the sialidase activity of the first fusion protein.

[0155] In some embodiments, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 15%, about 1% to about 10%, about 5% to about 10%, about 2% to about 15%, about 3% to about 15%, about 4% to about 15%, or about 5% to about 15%. In some embodiments, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 15%. In some embodiments, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 14%.

[0156] In some embodiments, the weight percentage of the third subspecies in the total protein in the composition is at least or at least about any one of 1%, 1.2%, 1.3%, 1.5%, 2%, or 3%. In some embodiments, the weight percentage of the third subspecies in the total protein in the composition is at least or at least about 1%. In some embodiments, the weight percentage of the third subspecies in the total protein in the composition is at least or at least about 3%.

[0157] In some embodiments, the weight percentage of the third subspecies of the total protein in the composition is at most or at most about any one of 30%, 20%, 15%, or 10%. In some embodiments, the weight percentage of the third subspecies of the total protein in the composition is at most about 14%.

[0158] In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the third subspecies elutes at about 56-59 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the third subspecies elutes at about 57-58 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the third subspecies elutes at about 57.5 mM CaCl2.

[0159] In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 5% or about 4% to about 5%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 5% or about 4% to about 5%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 5% or about 4% to about 5%. In some embodiments, after storage at room temperature for 3 days (e.g., 70 to 74 hours), the weight percentage of the third subspecies in the total protein contained in the composition is about 1% to about 5% or about 4% to about 5%. In some embodiments, after storage at room temperature for about 7 days (e.g., 166-170 hours), the weight percentage of the third subspecies of the total protein in the composition is about 1% to about 5% or about 4% to about 5%.

[0160] In some embodiments, after storage at room temperature, the weight percentage of the third subspecies in the total protein of the composition is at least about any one of 1%, 2%, 3%, 5%, 10%, or more. In some embodiments, after storage at room temperature, the weight percentage of the third subspecies in the total protein of the composition is at most about any one of 30%, 20%, 15%, 14%, 12%, or 10%. In some embodiments, after storage at room temperature, the weight percentage of the third subspecies in the total protein of the composition is at most about any one of 1%-5%, 5%-10%, 10%-15%, 15%-20%, or 20%-30%. In some embodiments, after storage at room temperature, the weight percentage of the third subspecies in the total protein of the composition increases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, after storage at room temperature, the weight percentage of the third subspecies in the total protein in the composition is reduced by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, the weight percentage is measured after at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more storage at room temperature. In some embodiments, the weight percentage is measured after at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours storage at room temperature. In some embodiments, the weight percentage is measured after 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours-48 hours, 48 ​​hours-7 days, or 6 hours-7 days storage at room temperature.

[0161] In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43539.50 Da and 43559.50 Da. In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43544.50 Da and 43554.50 Da. In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43545.50 Da and 43553.50 Da. In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43546.50 Da and 43552.50 Da. In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43547.50 Da and 43551.50 Da. In some embodiments, the third subspecies exhibits a first peak having a mass between 43548.50 Da and 43550.50 Da by intact mass analysis.

[0162] In some embodiments, the third subspecies exhibits a second peak having a mass by intact mass analysis between 43671.20 Da and 43691.20 Da. In some embodiments, the third subspecies exhibits a second peak having a mass by intact mass analysis between 43676.20 Da and 43686.20 Da. In some embodiments, the third subspecies exhibits a second peak having a mass by intact mass analysis between 43677.20 Da and 43685.20 Da. In some embodiments, the third subspecies exhibits a second peak having a mass by intact mass analysis between 43678.20 Da and 43684.20 Da. In some embodiments, the third subspecies exhibits a second peak having a mass by intact mass analysis between 43679.20 Da and 43683.20 Da. In some embodiments, the third subspecies exhibits a second peak having a mass between 43680.20 Da and 43682.20 Da by intact mass analysis.

[0163] In some embodiments, the third subspecies exhibits a third peak having a mass by intact mass analysis between 43717.50 Da and 43737.50 Da. In some embodiments, the third subspecies exhibits a third peak having a mass by intact mass analysis between 43722.50 Da and 43732.50 Da. In some embodiments, the third subspecies exhibits a third peak having a mass by intact mass analysis between 43723.50 Da and 43731.50 Da. In some embodiments, the third subspecies exhibits a third peak having a mass by intact mass analysis between 43724.50 Da and 43730.50 Da. In some embodiments, the third subspecies exhibits a third peak having a mass by intact mass analysis between 43725.50 Da and 43729.50 Da. In some embodiments, the third subspecies exhibits a third peak having a mass of 43726.50 Da to 43728.50 Da by intact mass analysis.

[0164] In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43539.50 Da and 43559.50 Da, a second peak having a mass by intact mass analysis between 43671.20 Da and 43691.20 Da, and a third peak having a mass by intact mass analysis between 43717.50 Da and 43737.50 Da.

[0165] In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43544.50 Da and 43554.50 Da, a second peak having a mass by intact mass analysis between 43676.20 Da and 43686.20 Da, and a third peak having a mass by intact mass analysis between 43722.50 Da and 43732.50 Da.

[0166] In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43545.50 Da and 43553.50 Da, a second peak having a mass by intact mass analysis between 43677.20 Da and 43685.20 Da, and a third peak having a mass by intact mass analysis between 43723.50 Da and 43731.50 Da.

[0167] In some embodiments, the third subspecies exhibits a first peak having a mass by intact mass analysis between 43546.50 Da and 43552.50 Da, a second peak having a mass by intact mass analysis between 43678.20 Da and 43684.20 Da, and a third peak having a mass by intact mass analysis between 43724.50 Da and 43730.50 Da.

[0168] D. The fourth subspecies The fourth subspecies comprises a dimerized form of the first fusion protein. In some embodiments, the first fusion protein comprises a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0169] In some embodiments, the fourth subspecies comprises a methionine oxidized form of the first fusion protein. In some embodiments, the first fusion protein comprises a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1. In some embodiments, the sialidase domain comprises amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein comprises amino acids 1-415 of SEQ ID NO:1.

[0170] In some embodiments, the protein in the fourth subspecies has at least or at least about 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 40% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 45% of the sialidase activity of the first fusion protein. In some embodiments, the protein in the second subspecies has at least or at least about 90% of the sialidase activity of the first fusion protein.

[0171] In some embodiments, the fourth subspecies comprises any of about 40% dimer and about 60% monomer, about 35% dimer and about 65% monomer, about 30% dimer and about 70% monomer, about 25% dimer and about 75% monomer, about 20% dimer and about 80% monomer, about 15% dimer and about 85% monomer, or about 10% dimer and about 90% monomer, hi some embodiments, the fourth subspecies comprises about 35% dimer and about 65% monomer.

[0172] In some embodiments, the fourth subspecies comprises a methionine oxidized form of the first fusion protein. The methionine oxidized form of the fourth subspecies of the first fusion protein comprises, for example, any number, for example, one or all, or a combination thereof, of oxidized methionines. In some embodiments, at least or at least any one of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% by weight of the fourth subspecies is the methionine oxidized form of the first fusion protein.

[0173] In some embodiments, 20% to 55%, 20% to 45%, 20% to 30%, 30% to 40%, 40% to 45%, 40% to 55%, 30% to 55%, or 35% to 55% by weight of the fourth subspecies is a methionine-oxidized form of the first fusion protein.

[0174] In some embodiments, at most or at most about 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% by weight of the fourth subspecies is a methionine-oxidized form of the first fusion protein.

[0175] In some embodiments, the weight percentage of the fourth subspecies of the total protein in the composition is between about 0.1% and about 15%, between about 0.1% and about 10%, between about 0.1% and about 5%, between about 0.1% and about 4%, between about 0.2% and about 4%, between about 0.3% and about 4%, between about 0.4% and about 4%, between about 0.5% and about 4%, between about 0.6% and about 4%, between about 0.7% and about 4%, between about 0.8% and about 4%, between about 0.9% and about 4%, between about 1% and about 4%, between about 2% and about 4%, between about 0.2% and about 3%, between about 0.3% to about 3%, about 0.4% to about 3%, about 0.5% to about 3%, about 0.6% to about 3%, about 0.7% to about 3%, about 0.8% to about 3%, about 0.9% to about 3%, about 1% to about 3%, 2% to about 3%, about 0.1% to about 2%, about 0.2% to about 2%, about 0.3% to about 2%, about 0.4% to about 2%, about 0.5% to about 2%, about 0.6% to about 2%, about 0.7% to about 2%, about 0.8% to about 2%, about 0.9% to about 2%, or about 1% to about 2%. In some embodiments, the weight percentage of the fourth subspecies in the total protein contained in the composition is about 0.1% to about 4%.

[0176] In some embodiments, the weight percentage of the fourth subspecies in the total protein of the composition is at least or at least about any one of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, or 1.3%. In some embodiments, the weight percentage of the fourth subspecies in the total protein of the composition is at least or at least about 0.5%. In some embodiments, the weight percentage of the fourth subspecies in the total protein of the composition is at least or at least about 1%.

[0177] In some embodiments, the weight percentage of the fourth subspecies in the total protein in the composition is at most or at most about any one of 20%, 15%, 10%, 5%, 4%, or 3%. In some embodiments, the weight percentage of the fourth subspecies in the total protein in the composition is at most about 10%. In some embodiments, the weight percentage of the fourth subspecies in the total protein in the composition is at most about 4%.

[0178] In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fourth subspecies elutes at about 180-200 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fourth subspecies elutes at about 185-200 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fourth subspecies elutes at about 190-200 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fourth subspecies elutes at about 195-200 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fourth subspecies elutes at about 200 mM CaCl2.

[0179] In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the fourth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the fourth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the fourth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 3 days (e.g., 70 to 74 hours), the weight percentage of the fourth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 7 days (e.g., 166-170 hours), the weight percentage of the fourth subspecies of the total protein in the composition is about 1% to about 5% or about 1% to about 3%.

[0180] In some embodiments, after storage at room temperature, the weight percentage of the fourth subspecies in the total protein in the composition is at least about any one of 1%, 2%, 3%, 5%, 10%, or more. In some embodiments, after storage at room temperature, the weight percentage of the fourth subspecies in the total protein in the composition is at most about any one of 30%, 20%, 15%, 14%, 12%, or 10%. In some embodiments, after storage at room temperature, the weight percentage of the fourth subspecies in the total protein in the composition is at most about any one of 1%-5%, 5%-10%, 10%-15%, 15%-20%, or 20%-30%. In some embodiments, after storage at room temperature, the weight percentage of the fourth subspecies in the total protein of the composition increases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, after storage at room temperature, the weight percentage of the fourth subspecies in the total protein of the composition decreases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, the weight percentage is measured after storage at room temperature for at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the weight percentage is measured after storage at room temperature for at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours. In some embodiments, the weight percentage is measured after storage at room temperature for 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours-48 hours, 48 ​​hours-7 days, or 6 hours-7 days.

[0181] In some embodiments, the fourth subspecies exhibits a first peak having a mass by intact mass analysis between 44647.42 Da and 44667.42 Da. In some embodiments, the fourth subspecies exhibits a first peak having a mass by intact mass analysis between 44652.42 Da and 44662.42 Da. In some embodiments, the fourth subspecies exhibits a first peak having a mass by intact mass analysis between 44653.42 Da and 44661.42 Da. In some embodiments, the fourth subspecies exhibits a peak having a mass by intact mass analysis between 44654.42 Da and 44660.42 Da. In some embodiments, the fourth subspecies exhibits a peak having a mass by intact mass analysis between 44654.92 Da and 44659.92 Da.

[0182] In some embodiments, the fourth subspecies exhibits a second peak having a mass by intact mass analysis between 44663.42 Da and 44683.42 Da. In some embodiments, the fourth subspecies exhibits a second peak having a mass by intact mass analysis between 44668.42 Da and 44678.42 Da. In some embodiments, the fourth subspecies exhibits a second peak having a mass by intact mass analysis between 44669.42 Da and 44677.42 Da. In some embodiments, the fourth subspecies exhibits a second peak having a mass by intact mass analysis between 44670.42 Da and 44676.42 Da. In some embodiments, the fourth subspecies exhibits a second peak having a mass by intact mass analysis between 44670.92 Da and 44675.92 Da. In some embodiments, the second peak of the fourth species comprises a methionine oxidation variant of the first peak of the fourth species. In some embodiments, the second peak has a mass 15-17 Da greater than the mass of the first peak. In some embodiments, the second peak has a mass about 16 Da greater than the mass of the first peak.

[0183] In some embodiments, the fourth subspecies exhibits a third peak having a mass by intact mass analysis of 44825.42 Da to 44845.42 Da. In some embodiments, the fourth subspecies exhibits a third peak having a mass by intact mass analysis of 44830.42 Da to 44840.42 Da. In some embodiments, the fourth subspecies exhibits a third peak having a mass by intact mass analysis of 44831.42 Da to 44839.42 Da. In some embodiments, the third peak comprises a gluconate adduct of the first peak. In some embodiments, the third peak has a mass 176-180 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass 177-179 Da greater than the mass of the first peak. In some embodiments, the third peak has a mass about 178 Da greater than the mass of the first peak.

[0184] E. The fifth subspecies In some embodiments, the compositions provided herein may optionally include a fifth subspecies. The fifth subspecies includes a dehydrated form of the first fusion protein. In some embodiments, the dehydrated form of the first fusion protein includes a loss of at least one or more water molecules, e.g., a loss of association, compared to a fully hydrated form of the fifth subspecies. In some embodiments, the first fusion protein includes a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain including amino acids 395-415 of SEQ ID NO:1. In some embodiments, the sialidase domain includes amino acids 1-394 of SEQ ID NO:1. In some embodiments, the first fusion protein includes amino acids 1-415 of SEQ ID NO:1.

[0185] In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 15%, about 1% to about 3%, about 2% to about 3%, about 5% to about 15%, about 10% to 15%, about 5% to about 10%, or about 1% to about 10%. In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 10%. In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 9%. In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 2% to about 9%.

[0186] In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition is at least or at least about any one of 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%. In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition is at least or at least about 0.5%. In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition is at least or at least about 1%.

[0187] In some embodiments, the weight percentage of the fifth subspecies of the total protein in the composition is at most or at most about any one of 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%. In some embodiments, the weight percentage of the fifth subspecies of the total protein in the composition is at most about 10%.

[0188] In some embodiments, after storage at room temperature for 6 to 8 hours, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 23 to 25 hours, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 47 to 49 hours, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 3 days (e.g., 70 to 74 hours), the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 5% or about 1% to about 3%. In some embodiments, after storage at room temperature for 7 days (e.g., 166 to 170 hours), the weight percentage of the fifth subspecies of the total protein in the composition is about 1% to about 5% or about 1% to about 3%.

[0189] In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition after storage at room temperature is at least about any one of 1%, 2%, 3%, 5%, 10%, or more. In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition after storage at room temperature is at most about any one of 30%, 20%, 15%, 14%, 12%, or 10%. In some embodiments, the weight percentage of the fifth subspecies in the total protein of the composition after storage at room temperature is at most about any one of 1%-5%, 5%-10%, 10%-15%, 15%-20%, or 20%-30%. In some embodiments, after storage at room temperature, the weight percentage of the fifth subspecies in the total protein of the composition increases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, after storage at room temperature, the weight percentage of the fifth subspecies in the total protein of the composition decreases by at most about any one of 1%, 2%, 3%, 5%, 10%, 15%, or 20% compared to before storage at room temperature. In some embodiments, the weight percentage is measured after storage at room temperature for at least about any one of 6 hours, 8 hours, 12 hours, 24 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the weight percentage is measured after storage at room temperature for at most about any one of 7 days, 6 days, 5 days, 4 days, 3 days, 48 ​​hours, 24 hours, 12 hours, 8 hours, or 6 hours. In some embodiments, the weight percentage is measured after storage at room temperature for 6-8 hours, 23-25 ​​hours, 47-49 hours, 3-7 days, 6 hours-48 hours, 48 ​​hours-7 days, or 6 hours-7 days.

[0190] In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fifth subspecies elutes at about 90-110 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fifth subspecies elutes at about 95-105 mM CaCl2. In some embodiments, when the composition is subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluted with a gradient containing up to 200 mM CaCl2 in an acetate buffer, the fifth subspecies elutes at about 100 mM CaCl2.

[0191] In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 0.5% to about 10%, about 1% to about 3%, about 2% to about 3%, about 5% to about 15%, about 10% to 15%, about 5% to about 10%, or about 1% to about 10%, indicating that the composition is suitable for human medical use. In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 9%, indicating that the composition is suitable for human medical use. In some embodiments, the weight percentage of the fifth subspecies in the total protein contained in the composition is about 1% to about 8%, indicating that the composition is suitable for human medical use.

[0192] 2. Preparation In some aspects, the compositions provided herein are provided in a lyophilized microparticle or lyophilized cake formulation, or in a liquid formulation reconstituted from a lyophilized formulation. In some embodiments, the liquid formulation is provided in a nebulizer, for example, a vibrating mesh nebulizer. In some embodiments, the compositions provided herein are provided in a dry powder formulation. In some embodiments, the compositions provided herein are provided in a lyophilized formulation (e.g., lyophilized microparticle powder or lyophilized cake) for liquid reconstitution. In some embodiments, a liquid composition reconstituted from a lyophilized formulation is provided.

[0193] A. Lyophilized Cake Formulation for Liquid Reconstitution In some aspects, provided herein are lyophilized formulations (e.g., lyophilized cakes) of a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some aspects, provided herein are lyophilized cake formulations of a composition comprising DAS181 or at least four subspecies of DAS181 as described in Section II.1 above. In some embodiments, provided herein are pharmaceutical formulations comprising a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain, b) about 95-98% w / w trehalose, c) about 0.2-0.4% w / w MgSO4, d) about 0.4-0.6% w / w sodium acetate, and e) about 0.1-0.3% w / w acetic acid. Unless otherwise stated, the weight percentages provided in Section II.2.B represent the dry weight percentages present in the lyophilized cake.

[0194] In some embodiments, the formulation comprises from about 0.5% w / w to about 10% w / w, from about 0.5% w / w to about 9% w / w, from about 0.5% w / w to about 8% w / w, from about 0.5% w / w to about 7% w / w, from about 0.5% w / w to about 6% w / w, from about 0.5% w / w to about 5% w / w, from about 0.5% w / w to about 4% w / w, 0.5% w / w to about 3% w / w, or from about 0.5% w / w to about 2% w / w of active agent.

[0195] In some embodiments, the formulation comprises about 1% to about 10% w / w of active agent, about 1% to about 9% w / w, about 1% to about 8% w / w, about 1% to about 7% w / w, about 1% to about 6% w / w, about 1% to about 5% w / w, about 1% to about 4% w / w of active agent, about 1% to about 3% w / w of active agent, about 1% to about 2% w / w of active agent, or about 1.2% to 1.4% w / w of active agent. In some embodiments, the formulation comprises any one of about 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% w / w of active agent. In some embodiments, the formulation comprises 1.2% w / w to 1.4% w / w of active agent. In some embodiments, the formulation comprises about 1.3% w / w of active agent. In some embodiments, the formulation comprises 1.3% w / w of active agent.

[0196] In some embodiments, the formulation comprises at least about any one of 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% w / w of active agent. In some embodiments, the formulation comprises at least 1.1% w / w of active agent.

[0197] In some embodiments, the formulation comprises at most or at most about any one of 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% w / w of active agent. In some embodiments, the formulation comprises at most 3% w / w of active agent, at most 4% w / w of active agent, at most 5% w / w of active agent, or at most 10% w / w of active agent.

[0198] In some embodiments, the active agent is DAS181, the sequence of which is set forth in SEQ ID NO:1 or SEQ ID NO:2 (without the amino-terminal methionine). In some embodiments, the active agent is A) a polypeptide comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:1, B) a polypeptide comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:2, or C) a mixture of a polypeptide comprising (or consisting of, or consisting essentially of) SEQ ID NO:1 and a polypeptide comprising (or consisting of, or consisting essentially of) SEQ ID NO:2.

[0199] In some embodiments, the active agent is a mixture of DAS181 and its variants, including at least four subspecies as described in Section II.1 above. In some embodiments, the active agent comprises or consists of a mixture of DAS181 that can be separated by CEX-HPLC, the formulation comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid position being relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via the C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein. In some embodiments, the mixture further comprises a fifth subspecies of DAS181 described in Section II.1 above.

[0200] In some embodiments, the formulation comprises trehalose at about 70% w / w to about 99% w / w, about 75% w / w to about 99% w / w, about 80% w / w to about 99% w / w, about 85% w / w to about 99% w / w, about 90% w / w to about 99% w / w, about 95% w / w to about 99% w / w, 95% w / w to 99% w / w, about 95% w / w to about 98%, 95% w / w to 98% w / w, 96% w / w to 99% w / w, or 97% w / w to 99% w / w. In some embodiments, the formulation comprises trehalose at about 97.5% w / w or trehalose at 97.5% w / w.

[0201] In some embodiments, the formulation comprises at least or at least about any one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, or 97% w / w trehalose. In some embodiments, the formulation comprises at least or at least about 95% w / w trehalose. In some embodiments, the formulation comprises at least or at least about 97% w / w trehalose.

[0202] In some embodiments, the formulation comprises trehalose at or about any one of at most 99%, 98%, or 97.5% w / w.

[0203] In some embodiments, the formulation comprises any one of about 0.15% w / w to about 0.45% w / w, about 0.2% w / w to about 0.4% w / w, or about 0.25% w / w to about 0.35% w / w MgSO4. In some embodiments, the formulation comprises 0.25% w / w to 0.35% w / w MgSO4. In some embodiments, the formulation comprises about 0.3% w / w MgSO4 or 0.3% w / w MgSO4.

[0204] In some embodiments, the formulation comprises at least or at least about any one of 0.1%, 0.15%, 0.2%, 0.25%, or 0.3% w / w MgSO4. In some embodiments, the formulation comprises at least or at least about 0.2% w / w MgSO4.

[0205] In some embodiments, the formulation comprises at most or at most about any one of 3%, 2%, 1%, 0.7%, 0.5%, 0.4%, or 0.3% w / w MgSO4. In some embodiments, the formulation comprises at most about 0.5% w / w MgSO4.

[0206] In some embodiments, the formulation comprises any one of about 0.3% w / w to about 0.7% w / w, 0.4% w / w to about 0.6% w / w, or about 0.45% w / w to about 0.55% w / w of sodium acetate. In some embodiments, the formulation comprises 0.45% w / w to 0.55% w / w of sodium acetate. In some embodiments, the formulation comprises about 0.5% w / w or 0.5% w / w of sodium acetate.

[0207] In some embodiments, the formulation comprises at least or at least about 0.1% w / w sodium acetate, at least or at least about 0.2% w / w sodium acetate, at least or at least about 0.25% w / w sodium acetate, at least or at least about 0.3% w / w sodium acetate, at least or at least about 0.4% w / w sodium acetate, or at least or at least about 0.5% w / w sodium acetate. In some embodiments, the formulation comprises at least or at least about 0.3% w / w sodium acetate.

[0208] In some embodiments, the formulation comprises at most about 2% sodium acetate w / w, at most about 1% sodium acetate w / w, at most about 0.8% sodium acetate w / w, at most about 0.7% sodium acetate w / w, at most about 0.6% sodium acetate w / w, or at most about 0.5% sodium acetate w / w. In some embodiments, the formulation comprises at most about 0.7% sodium acetate w / w.

[0209] In some embodiments, the formulation has a pH of about 4.5 to about 6.5, about 5.4 to about 6.4, or about 5 to about 6. In some embodiments, the formulation has a pH of 5 to 6. In some embodiments, the formulation has a pH of 5.3 or about 5.3.

[0210] In some embodiments, the formulation has at least or at least about any one of pH 4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0. In some embodiments, the formulation has at least or at least about pH 4.7.

[0211] In some embodiments, the formulation has a pH of at most about any one of 7.0, 6.5, 6.3, 6.0, 5.9, 5.8, or 5.7. In some embodiments, the formulation has a pH of at most about 6.5. In some embodiments, the formulation has a pH of at most about 6.3.

[0212] In some embodiments, the formulation comprises a buffering agent. In some embodiments, the buffering agent has a pH of about 4.5 to about 6.5, about 5.4 to about 6.4, or about 5 to about 6. In some embodiments, the buffering agent is acetic acid. In some embodiments, the formulation comprises about 0.1% w / w to about 0.3% w / w acetic acid or about 0.15% w / w to about 0.25% w / w acetic acid. In some embodiments, the formulation comprises 0.2% or about 0.2% w / w acetic acid. In some embodiments, the formulation comprises at least or at least about any one of 0.05%, 0.1%, 0.15%, or 0.2% w / w acetic acid. In some embodiments, the formulation comprises at least or at least about 0.1% w / w acetic acid. In some embodiments, the formulation comprises at most or at most about any one of 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, or 0.2% w / w acetic acid. In some embodiments, the formulation comprises at most about 0.5% w / w acetic acid.

[0213] In some embodiments, the formulation does not include histidine. In some embodiments, the formulation does not include CaCl2.

[0214] In some embodiments, provided herein is a reconstituted liquid formulation produced by reconstituting a lyophilized cake formulation in water. In some embodiments, the reconstituted liquid formulation comprises about 70 mg / mL to about 100 mg / mL, about 75 mg / mL to about 100 mg / mL, about 80 mg / mL to about 100 mg / mL, about 85 mg / mL to about 100 mg / mL, or about 90 mg / mL to about 100 mg / mL of trehalose. In some embodiments, the reconstituted liquid formulation comprises trehalose at about 91 mg / mL to about 100 mg / mL, about 92 mg / mL to about 100 mg / mL, about 93 mg / mL to about 100 mg / mL, about 90 mg / mL to about 99 mg / mL, about 90 mg / mL to about 98 mg / mL, about 90 mg / mL to about 97 mg / mL, about 90 mg / mL to about 96 mg / mL, about 90 mg / mL to about 95 mg / mL, or about 92 mg / mL to about 96 mg / mL. In some embodiments, the reconstituted liquid formulation comprises trehalose at 92 mg / mL to 96 mg / mL. In some embodiments, the reconstituted liquid formulation comprises trehalose at 93 mg / mL to 95 mg / mL. In some embodiments, the reconstituted liquid formulation comprises trehalose at about 94 mg / mL. In some embodiments, the reconstituted liquid formulation comprises 94 mg / mL trehalose.

[0215] In some embodiments, the reconstituted liquid formulation comprises at least or at least about any one of 70, 80, 90, 91, 92, 93, or 94 mg / mL trehalose. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 90 mg / mL trehalose. In some embodiments, the reconstituted liquid formulation comprises at most or at most about any one of 100, 99, 98, 97, 96, or 95 mg / mL trehalose. In some embodiments, the reconstituted liquid formulation comprises at most about 96 mg / mL trehalose.

[0216] In some embodiments, the reconstituted liquid formulation comprises about 0.1 mg / mL to about 0.3 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises 0.1 mg / mL to 0.3 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises about 0.15 mg / mL to about 0.25 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises 0.15 mg / mL to 0.25 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises about 0.2 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises 0.2 mg / mL of acetic acid.

[0217] In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.05 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.1 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.15 mg / mL of acetic acid. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.2 mg / mL of acetic acid.

[0218] In some embodiments, the reconstituted liquid formulation comprises at most about 1 mg / mL acetic acid, at most about 0.8 mg / mL acetic acid, at most about 0.7 mg / mL acetic acid, at most about 0.6 mg / mL acetic acid, at most about 0.5 mg / mL acetic acid, at most about 0.4 mg / mL acetic acid, or at most about 0.3 mg / mL acetic acid. In some embodiments, the reconstituted liquid formulation comprises at most about 0.4 mg / mL acetic acid.

[0219] In some embodiments, the reconstituted liquid formulation comprises sodium acetate at about 0.4 mg / mL to about 0.6 mg / mL, about 0.45 mg / mL to about 0.55 mg / mL, or 0.45 mg / mL to 0.55 mg / mL. In some embodiments, the reconstituted liquid formulation comprises sodium acetate at about 0.5 mg / mL or about 0.5 mg / mL.

[0220] In some embodiments, the reconstituted liquid formulation comprises at least or at least about any one of 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL of sodium acetate. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.3 mg / mL of sodium acetate. In some embodiments, the reconstituted liquid formulation comprises at most or at most about any one of 1, 0.8, 0.9, 0.7, or 0.6 mg / mL of sodium acetate. In some embodiments, the reconstituted liquid formulation comprises at most about 0.7 mg / mL of sodium acetate.

[0221] In some embodiments, the reconstituted liquid formulation comprises about 0.2 mg / mL to about 0.5 mg / mL MgSO4 or about 0.2 mg / mL to about 0.4 mg / mL MgSO4. In some embodiments, the reconstituted liquid formulation comprises about 0.3 mg / mL MgSO4. In some embodiments, the reconstituted liquid formulation comprises 0.3 mg / mL MgSO4. In some embodiments, the formulation does not comprise histidine or CaCl2.

[0222] In some embodiments, the reconstituted liquid formulation comprises at least or at least about any one of 0.05, 0.1, 0.15, 0.2, or 0.3 mg / mL MgSO4. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 0.1 mg / mL MgSO4.

[0223] In some embodiments, the reconstituted liquid formulation comprises at most or at most about any one of 1, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4 mg / mL of MgSO4. In some embodiments, the reconstituted liquid formulation comprises at most about 0.5 mg / mL of MgSO4.

[0224] In some embodiments, the reconstituted liquid formulation comprises at or about 0.5 mg / mL to 5 mg / mL, at or about 0.5 mg / mL to 4 mg / mL, at or about 0.5 mg / mL to 3 mg / mL, at or about 0.5 mg / mL to 2 mg / mL, at or about 1 mg / mL to 1.5 mg / mL, at or about 1.1 mg / mL to 2 mg / mL, or at or about 1.2 mg / mL to 2 mg / mL of active agent. In some embodiments, the reconstituted liquid formulation comprises at or about 0.9, 1, 1.1, 1.2, or 1.3 mg / mL of active agent. In some embodiments, the reconstituted liquid formulation comprises at least or at least about 1 mg / mL of active agent. In some embodiments, the reconstituted liquid formulation comprises at most or at most about any one of 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, or 1.4 mg / mL of active agent. In some embodiments, the reconstituted liquid formulation comprises at most about 1.8 mg / mL of active agent.

[0225] In some embodiments, the reconstituted liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the reconstituted liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the reconstituted liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0226] In some embodiments, provided herein is a pharmaceutical formulation comprising, by dry weight, a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain, b) about 95-98% w / w trehalose, c) about 0.2-0.4% w / w MgSO4, d) about 0.4-0.6% w / w sodium acetate, and e) about 0.1-0.3% w / w acetic acid. In some embodiments, the pharmaceutical formulation comprises, by dry weight, about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid. In some embodiments, the pharmaceutical formulation is a liquid formulation reconstituted from a lyophilized formulation. In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid. In some embodiments, the fusion protein is DAS181, the sequence of which is set forth in SEQ ID NO:1 (with an amino-terminal methionine) or SEQ ID NO:2 (without an amino-terminal methionine). In some embodiments, the fusion protein is A) a polypeptide comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:1, B) a polypeptide comprising (or consisting of, or consisting essentially of) the amino acid sequence of SEQ ID NO:2, or C) a mixture of a polypeptide comprising (or consisting of, or consisting essentially of) SEQ ID NO:1 and a polypeptide comprising (or consisting of, or consisting essentially of) SEQ ID NO:2.

[0227] In some embodiments, provided herein are: i) a first subspecies comprising a) a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid positions being relative to SEQ ID NO:1; c) a second subspecies comprising a sialidase domain fused via its C-terminus to a truncated cationic domain. a) a mixture of DAS181 subspecies separable by CEX-HPLC, comprising a second fusion protein comprising a truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1, and b) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain, ii) about 95-98% w / w trehalose, iii) about 0.2-0.4% w / w MgSO4, iv) about 0.4-0.6% w / w sodium acetate, and v) about 0.1-0.3% w / w acetic acid, by dry weight. In some embodiments, the pharmaceutical formulation comprises, by dry weight, about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid. In some embodiments, the pharmaceutical formulation is a liquid formulation reconstituted from a lyophilized formulation. In some embodiments, the liquid formulation comprises 1-2 mg / mL of DAS181 and DAS181 variant subspecies, 10-100 mg / mL of trehalose, 0.1-0.5 mg / mL of MgSO4, 0.2-0.7 mg / mL of sodium acetate, and 0.1-0.3 mg / mL of acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL of DAS181 protein (including subspecies), about 94 mg / mL of trehalose, about 0.3 mg / mL of MgSO4, about 0.5 mg / mL of sodium acetate, and about 0.2 mg / mL of acetic acid.In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation is provided in a nebulizer, for example a vibrating mesh nebulizer.

[0228] In some aspects, provided herein is a nebulizer comprising a liquid formulation comprising a composition comprising DAS181 or a plurality of DAS181 subspecies or variants thereof. In some embodiments, the nebulizer is a vibrating mesh nebulizer. In some embodiments, the liquid formulation comprises 1-2 mg / mL of DAS181 and DAS181 variant subspecies, 10-100 mg / mL of trehalose, 0.1-0.5 mg / mL of MgSO4, 0.2-0.7 mg / mL of sodium acetate, and 0.1-0.3 mg / mL of acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL of DAS181 protein (including subspecies), about 94 mg / mL of trehalose, about 0.3 mg / mL of MgSO4, about 0.5 mg / mL of sodium acetate, and about 0.2 mg / mL of acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid. In some embodiments, the subspecies of DAS181 and DAS181 variants comprise a mixture of subspecies of DAS181 that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain.

[0229] B. Lyophilized Microparticle Formulations In some embodiments, a formulation is provided that comprises at least four subspecies of DAS181 that can be separated by CEX-HPLC, the formulation comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid position relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via the C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein, the formulation being formulated into a microparticle formulation. Suitable lyophilized microparticle formulations are described in US 9,700,602, the contents of which are incorporated herein by reference in their entirety.

[0230] In some embodiments, a formulation comprising at least four subspecies of DAS181 is provided in the following microparticle formulation (also referred to herein as "Formulation II - non-anhydrous"): a) DAS181: 64.5-64.7% (w / w%) of total protein containing at least four DAS181 variants as described in Section II.1 above -b) Bases not containing histidine: 4.3 to 4.6% (w / w%) -c) Histidine HCl: 5.8-6.3% (w / w%) -d) Trehalose: 9.0-9.7% (w / w%) -e) Magnesium sulfate: 4.6-5.9% (w / w%) -g) Water: 10.0% (w / w%, depends on humidity of storage conditions) In some embodiments, the formulation may also contain small amounts of sodium acetate (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, e.g., 0.03% w / w), small amounts of calcium chloride (less than 1% w / w, less than 0.5% w / w, less than 0.3% w / w, e.g., 0.3% w / w), and small amounts of acetic acid (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, e.g., 0.02% w / w). Small amounts of residual isopropanol may be present (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w).

[0231] In some embodiments, a formulation comprising at least four subspecies of DAS181 is provided in the following dry microparticle formulation (also referred to herein as "Formulation II - Anhydrous"): a) DAS181: 71.7-71.9% (w / w%) of total protein containing at least four DAS181 variants as described in Section II.1 above -b) Bases not containing histidine: 4.8 to 5.1% (w / w%) -c) Histidine HCl: 6.5-7.0% (w / w%) -d) Trehalose: 10.7 to 10.1% (w / w%) -e) Magnesium sulfate: 5.1-6.5% (w / w%) In some embodiments, the formulation may also include small amounts of sodium acetate (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, e.g., 0.03% w / w), small amounts of calcium chloride (less than 1% w / w, less than 0.5% w / w, less than 0.3% w / w, e.g., 0.3% w / w), and small amounts of acetic acid (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, e.g., 0.02% w / w). Small amounts of residual isopropanol may be present (less than 1% w / w, less than 0.5% w / w, less than 0.1% w / w, less than 0.05% w / w, less than 0.01% w / w). In some embodiments, the formulation does not include citric acid.

[0232] In one embodiment, the method for preparing Formulation II comprises the steps of: - (a) An excipient solution (pH 6) containing histidine, trehalose, magnesium sulfate and calcium chloride is prepared by mixing the stock solutions and sterile filtered. - (b) The excipient solution is added, with mixing, to a compounding vessel containing the sialidase fusion protein (total protein comprising at least four DAS181 subspecies as described in Section II.1 above) at an initial concentration of 125 mg / ml total protein. - (c) Sterile filtered isopropanol is added to the compounding vessel with stirring to form a stock solution. The final composition of the stock solution is as follows: 70 mg / ml sialidase fusion protein (total protein including at least four DAS181 subspecies described in Section II.1 above), 25% isopropanol, 4.99 mg / ml histidine, 6.80 mg / ml histidine-HCl, 10.50 mg / ml trehalose, 5.06 mg / ml magnesium sulfate, 0.21 mg calcium chloride, 0.05 mg / ml sodium acetate, 0.02 mg / ml acetic acid. The pH of the solution is 6.0. The time between the start of the isopropanol addition and the start of the lyophilization cycle is within 60 minutes. - (e) Each depyrogenated stainless steel tray is filled with 18 g of stock solution using a metering pump. - (f) Subject the filled stainless steel trays to the following freeze-drying cycle: -a. attach gaskets to the trays and place them on the shelves of a freeze dryer at 25°C for 5 minutes; -b. decreasing the temperature of the shelf to -45°C at a rate of -0.5°C / min; - c. Primary drying was achieved by setting the condenser to below -80°C, applying a vacuum of 125 mTorr, and increasing the temperature at a rate of 1°C / min to -0°C, then holding for 60 hours; - d. secondary drying is achieved by increasing the temperature to 30°C at a rate of 1°C / min and then holding for 6 hours; -e. Release the vacuum and backfill the lyophilizer with nitrogen to prevent oxidation of the microparticle formulation, then transfer to bulk mix bottles and divide the bulk powder into aliquots for storage at -15°C or below.

[0233] The microparticles of Formulation II may have one or more of the following: MMAD 6.5 microns (or 2-8 microns, 3-8 microns or 5-7), GSD 1.5-1.7 (or 1.3-1.9 or 1.4-1.8), FPF (volume % less than 5 microns) 6.6% (35%, 30%, 25%, 20%, 15%, 10% or 5%), and Tg 38°C.

[0234] Thus, in some embodiments, the microparticles have a MMAD of 3-8 (5-7) microns (6.2-6.8 microns), a GSD of 1.3-1.6 (1.4-1.6), an FPF of less than 9% (less than 8%, less than 7%, about 6-7%), and contain (by weight) sialidase fusion protein (total protein including at least four DAS181 variants described in Section II.1 above): 60-70% (62-68%, 64-66%, 65%), histidine free base: 3-6% (4-5%), histidine HCl: 5-9% (5-7%, 8-9%), trehalose: 7-11% (8-10%, 8.5-9.5%), magnesium sulfate: 4-8% (5-7%, 4-6%), and water: 6-12% (8-12%, 9-11%). The microparticles may also contain small amounts of sodium acetate (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%), small amounts of calcium chloride (less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, e.g., 0.1-0.3%), and small amounts of acetic acid (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.01%). Small amounts of residual isopropanol may be present (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%).

[0235] When the microparticles of Formulation II are anhydrous, they may contain (by weight) sialidase fusion protein (total protein including at least four DAS181 variants described in Section II.1 above): 69-74% (70-73%, 71-72%, 72%), histidine-free base: 3-8% (4-7%, 4-6%), histidine HCl: 4-9% (5-8%, 6-7%), trehalose: 8-12% (9-11%, 8-10%), magnesium sulfate: 4-8% (5-7%, 6-7%). The microparticles may also contain small amounts of sodium acetate (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%), small amounts of calcium chloride (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%), and small amounts of acetic acid (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.01%). Small amounts of residual isopropanol may be present (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%).

[0236] Microparticle Formulation I In some embodiments, the sialidase fusion protein (the whole protein comprising at least four subspecies of DAS181 as described in Section II.1 above) is provided in the following microparticle formulation (also referred to herein as "Formulation I - not anhydrous"): - a) Sialidase fusion protein: 86.7% (w / w%) of the total protein containing at least four DAS181 variants as described in section II.1 above -b) Sodium sulfate: 2.5% (w / w%) -c) Water: 10.0% (w / w%, depends on humidity of storage conditions) In some embodiments, the formulation may also include small amounts of sodium acetate (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%), small amounts of calcium chloride (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.03%), and small amounts of acetic acid (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, e.g., 0.01%). Small amounts of residual isopropanol may be present (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%). In some embodiments, a dry microparticle formulation (also referred to herein as "Formulation I - Anhydrous") is provided that includes: -a) Sialidase fusion protein: 96.3% (w / w%) of total protein containing at least four DAS181 variants as described in Section II.1 above -b) Sodium sulfate 2.7% (w / w%) The microparticles of Formulation I may also contain small amounts of sodium acetate (less than 1%, less than 0.8%, less than 0.7%, less than 0.6%), small amounts of calcium chloride (less than 1%, less than 0.5%, less than 0.4%), and small amounts of acetic acid (less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, e.g., 0.2%). Small amounts of residual isopropanol may be present (less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%).

[0237] The microparticles of Formulation I may have one or more of the following: MMAD 4-7 microns (or 2-8 microns, 3-8 microns or 5-7 microns), GSD 1.5-1.7 (or 1.3-1.9 or 1.4-1.8), FPF (volume % less than 5 microns) 8% (less than 35%, 30%, 25%, 20%, 15%, 10% or 5%), and Tg 38°C.

[0238] Thus, in some embodiments, the microparticles have a MMAD of 3-8 (5-7) microns (6.2-6.8 microns), a GSD of 1.3-1.6 (1.4-1.6), an FPF of less than 9% (less than 8%, less than 7%, about 6-7%), and contain (by weight) a sialidase fusion protein (total protein comprising at least four DAS181 subspecies as described in Section II.1 above).

[0239] The microparticle formulations obtained by the methods provided herein (including, for example, Formulation I and Formulation II) have a relatively uniform size distribution, i.e., are relatively monodisperse, with a geometric standard deviation (GSD) of about 1.2 to 2.0, typically about 1.2 to 1.5, 1.6, 1.7, or 1.8. The particles are also homogeneous, i.e., the components of the formulation are not separated but are uniformly distributed throughout the particle. Furthermore, the fine particle fraction (FPF), which comprises microparticles less than 5 microns, is less than 10%, typically less than about 8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, or 1%.

[0240] III. Methods for preparing and evaluating the composition In some aspects, the DAS181 compositions provided herein, including at least four DAS181 subspecies, can be prepared according to standard protein expression and purification methods. In some embodiments, the DAS181 compositions can be purified from cells that contain an expression vector encoding one or more of the DAS181 subspecies sequences. In some embodiments, the DAS181 compositions can be purified from cells that contain an expression vector encoding the amino acid sequence of SEQ ID NO:1. Overexpression of proteins in cells (e.g., bacterial cells) can be achieved using expression vectors. Expression vectors can be autonomous or integrative. Recombinant nucleic acids (e.g., encoding the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:1) can be introduced into cells in the form of expression vectors, such as plasmids. Recombinant nucleic acids can be maintained extrachromosomally or integrated into chromosomal DNA. Expression vectors can include a selectable marker gene encoding a protein required for cell survival under selected conditions (to allow for detection and / or selection of cells transformed with the desired nucleic acid). The expression vector can also contain an autonomously replicating sequence (ARS).

[0241] Transformed cells (i.e., microbial cells) may be selected using appropriate techniques, including, but not limited to, culturing auxotrophic cells after transformation in the absence of a required biochemical, selecting and detecting new phenotypes, or culturing in the presence of an antibiotic that is toxic to yeast in the absence of the resistance gene contained in the transformant. Transformants may also be selected and / or verified by integrating the expression cassette into the genome, which may be assessed, for example, by Southern blot or PCR analysis. Prior to introducing the vector into a target cell of interest, the vector may be propagated (e.g., amplified) in bacterial cells, such as Escherichia coli (E. coli) or Pseudomonas fluorescens (P. fluorescens), as described above. Pseudomonas fluorescens expression systems are described, for example, in U.S. Patent Nos. 10,041,102 and 8,288,127, the contents of each of which are incorporated herein by reference in their entirety. The vector DNA may be isolated from the bacterial cells by any method known in the art that results in the purification of the vector DNA from the bacterial environment. Because microbial proteins can be toxic to mammalian cells, the purified vector DNA is extensively extracted using phenol, chloroform, and ether to ensure that these proteins are not present in the plasmid DNA preparation.

[0242] Expression systems that can be used for small- or large-scale production of the polypeptides include, but are not limited to, microorganisms, such as bacteria (e.g., E. coli or P. fluorescens) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing the nucleic acid molecule, or fungi (e.g., S. cerevisiae) transformed with a recombinant fungal expression vector containing the nucleic acid molecule.

[0243] Generally, for in vivo production of a protein of interest by bacterial (e.g., E. coli or P. fluorescens) recombinant cells, the cells can be cultured (shaking culture, submerged culture, etc.) in an aqueous nutrient medium containing assimilable nitrogen and carbon sources, usually under submerged aerobic conditions. The aqueous medium can be maintained at a pH of 4.0 to 8.0 (e.g., 4.5, 5.0, 5.5, 6.0, or 7.5) using protein components contained in the medium, buffers incorporated in the medium, or by external addition of acids or bases as needed. Suitable carbon sources contained in the nutrient medium can include, for example, carbohydrates, lipids, and organic acids, such as glucose, sucrose, fructose, glycerol, starch, vegetable oils, oils derived from petrochemicals, succinic acid, formic acid, etc. Suitable nitrogen sources include, for example, yeast extract, corn steep liquor, meat extract, peptone, vegetable meal, distiller's grains, dried yeast, and the like, and inorganic nitrogen sources such as ammonium sulfate, ammonium phosphate, nitrates, urea, amino acids, and the like.

[0244] Advantageously, the carbon and nitrogen sources used in combination do not need to be used in pure form, since less pure materials containing trace amounts of growth factors and large amounts of inorganic nutrients are also suitable for use. Desired inorganic salts, such as sodium or potassium phosphate, sodium or potassium chloride, magnesium salts, copper salts, etc., can be added to the medium. If necessary, a trace amount of an antifoaming agent, such as liquid paraffin or vegetable oil, may be added, but is usually not necessary.

[0245] Cultivation of recombinant cells (e.g., E. coli or P. fluorescens cells) expressing a protein of interest can be performed under conditions that enhance optimal biomass and / or enzyme titer yields. Such conditions include, for example, batch, fed-batch, or continuous culture. Furthermore, the parameters of the conditions can also be modified to enhance optimal biomass and / or enzyme titer yields of the DAS181 protein. Such conditions include, for example, glycerol concentration and high pO2 in the medium. For large amounts of biomass production, submerged aerobic culture methods can be used, while small amounts can be cultured in shake flasks. For large tank production, multiple small inoculum tanks can be used to make the inoculum high enough to minimize lag time in the production vessel. The medium for the production of the biocatalyst is generally sterilized (e.g., by autoclaving) before inoculating the cells. Aeration and agitation of the culture can be achieved by mechanical means with simultaneous addition of sterile air, or by adding air alone in a bubble reactor. For example, during cultivation in a bioreactor, a higher pO2 (dissolved oxygen) can be used to increase optimal biomass. This can also be used to increase optimal active protein expression in the biomass culture. The introduction of such fermentation parameters, including higher oxygen tension and gradual glycerol depletion, can increase the production of the protein of interest.

[0246] In some embodiments, provided herein is a method of releasing a composition of DAS181 subspecies, such as any one of the compositions described herein, for human medical use, comprising subjecting the composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC. In some embodiments, the composition released for human medical use meets government regulatory standards, such as standards set by the U.S. FDA. In some embodiments, the CEX-HPLC includes using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acid, and eluting with a gradient containing up to 200 mM CaCl2 in acetate buffer. In some embodiments, provided herein is a pharmaceutical composition released for human medical use by the release method.

[0247] In some embodiments, provided herein is a method for evaluating the suitability of a composition of DAS181 for human medical use, comprising subjecting the composition to CEX-HPLC and determining the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC. In some embodiments, the CEX-HPLC comprises using non-porous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluting with a gradient of up to 200 mM CaCl2 in acetate buffer. In some embodiments, provided herein is a pharmaceutical composition identified as suitable for human medical use by the method for evaluating suitability. In some embodiments, the method comprises determining the weight percentages of the first, second, third, and fourth subspecies separated by the CEX-HPLC. In some embodiments, the method comprises deeming the composition of DAS181 suitable for human medical use if the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is at or about 30% or less, the weight percentage of the third subspecies of the total protein in the composition is at or about 15% or less, and the weight percentage of the fourth subspecies of the total protein in the composition is 4% or less.

[0248] In some embodiments, provided herein is a method of releasing a commercial batch of DAS181 for human medical use, the method comprising subjecting the composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC. In some embodiments, the CEX-HPLC comprises using non-porous 3 μm polystyrene divinylbenzene beads derivatized with a carboxylic acid and eluting with a gradient of up to 200 mM CaCl2 in acetate buffer. In some embodiments, provided herein is a commercial batch of DAS181 released by the published method. In some embodiments, provided herein is a pharmaceutical composition released by the published method for human medical use. In some embodiments, the method comprises releasing a commercial batch of DAS181 for human medical use when the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is at or about 30% or less, the weight percentage of the third subspecies of the total protein in the composition is at or about 15% or less, and the weight percentage of the fourth subspecies of the total protein in the composition is 4% or less.

[0249] In some embodiments, the percentage by weight of the first subspecies of the total protein in the composition is at least 50%, the percentage by weight of the second subspecies of the total protein in the composition is about 5% to about 30%, the percentage by weight of the third subspecies of the total protein in the composition is about 1% to about 15%, and the percentage by weight of the fourth subspecies of the total protein in the composition is about 0.1% to about 15%, indicating that the composition is suitable for medical use in humans.

[0250] In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is less than about 30%, the weight percentage of the third subspecies of the total protein in the composition is less than about 15%, and the weight percentage of the fourth subspecies of the total protein in the composition is less than about 15%, indicating that the composition is suitable for human medical use.

[0251] In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is less than about 30%, the weight percentage of the third subspecies of the total protein in the composition is less than about 15%, and the weight percentage of the fourth subspecies of the total protein in the composition is less than about 10%, indicating that the composition is suitable for human medical use.

[0252] In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is less than about 30%, the weight percentage of the third subspecies of the total protein in the composition is less than about 15%, and the weight percentage of the fourth subspecies of the total protein in the composition is less than about 5%, indicating that the composition is suitable for human medical use.

[0253] In some embodiments, the weight percentage of the first subspecies of the total protein in the composition is at least 50%, the weight percentage of the second subspecies of the total protein in the composition is at or about 30% or less, the weight percentage of the third subspecies of the total protein in the composition is at or about 15% or less, and the weight percentage of the fourth subspecies of the total protein in the composition is 4% or less, indicating that the composition is suitable for human medical use.

[0254] In some embodiments, an indication that the composition is suitable for medical use further includes a total protein concentration of 1.1-1.4 mg / g. In some embodiments, an indication that the composition is suitable for medical use further includes a monomer peak area percentage of at least 96% of the total protein in the sample as determined by size exclusion chromatography and / or SDS-PAGE analysis. In some embodiments, an indication that the composition is suitable for human medical use further includes a multimer peak area percentage of not more than 4% of the total protein in the sample as determined by size exclusion chromatography. In some embodiments, an indication that the composition is suitable for human medical use further includes a sialidase activity of 540-740 U / mg protein of the total protein in the composition. One milliunit (mU) of sialidase activity is defined as the amount of enzyme that releases 1.0 nmole of 4-Mu from MuNaNa in 20 minutes at 25° C. in 100 mmol glycine buffer containing 0.5% BSA at pH 8.5. In some embodiments, an indication that the composition is suitable for human medical use includes a solution reconstituted from the lyophilized composition being a clear, colorless solution with no visible residue or visible insoluble matter. In some embodiments, an indication that the composition is suitable for human medical use includes a solution reconstituted from the lyophilized composition having an osmolality of 270-330 mOsm / kg. In some embodiments, an indication that the composition is suitable for human medical use includes a solution reconstituted from the lyophilized composition having a pH of 4.5-6.5.In some embodiments, an indication that the composition is suitable for human medical use includes positive confirmation of a major band by SDS-PAGE that matches a reference standard, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid position relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-397 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein. In some embodiments, indications that the composition is suitable for human medical use include a relative Km and Kcat of 70% to 130% relative to the reference standard.

[0255] In some embodiments, determining whether the composition is suitable for human medical use includes determining whether a vial containing a lyophilized cake formulation of the composition is suitable for human medical use. In some embodiments, an indication that the vial contains a lyophilized cake formulation of the composition includes a DAS181 and DAS181 variant subspecies content of 4.5 mg to 5.5 mg per vial as measured by high performance liquid chromatography. In some embodiments, an indication that the composition is suitable for human medical use includes a moisture content of the lyophilized cake of 4% or less. In some embodiments, an indication that the composition is suitable for human medical use includes a bacterial endotoxin level of less than 4.0 EU / vial. In some embodiments, an indication that the composition is suitable for human medical use includes a number of particles with a diameter of 25 μM or more of 600 or less per vial. In some embodiments, an indication that the composition is suitable for human medical use includes a number of particles with a diameter of 10 μM or more of 6000 or less per vial.

[0256] In some aspects, provided herein are methods of making pharmaceutical compositions, including compositions comprising DAS181 as described herein, the methods comprising: a) introducing a nucleic acid encoding a protein of SEQ ID NO:1 into a bacterial host cell; b) expressing the protein encoded by the nucleic acid in the bacterial host cell; c) purifying the protein by chromatography to obtain a purified protein composition; and d) evaluating the suitability of the purified protein composition for human medical use, the evaluating comprising subjecting the purified protein composition to CEX-HPLC and identifying the relative amounts of the first, second, third, fourth, and optionally fifth subspecies separated by the CEX-HPLC. In some embodiments, the chromatography comprises cation exchange chromatography (e.g., CEX-HPLC). In some embodiments, the chromatography comprises hydrophobic interaction chromatography (HIC). In some embodiments, the methods further comprise formulating the purified protein composition and obtaining a pharmaceutical composition. In some embodiments, the step of formulating the purified protein composition is performed after the step of evaluating the suitability of the purified protein composition. In other embodiments, the step of formulating the purified protein composition is performed prior to the step of evaluating the suitability of the purified protein composition. In some aspects, provided herein are pharmaceutical compositions made according to the methods of making a pharmaceutical composition.

[0257] Methods for bacterial expression of sialidase fusion proteins and purification of the sialidase fusion proteins are described, for example, in US Pat. No. 10,351,828, the contents of which are incorporated herein by reference in their entirety.

[0258] IV. Pharmaceutical Compositions, Kits and Articles of Manufacture Further provided herein are pharmaceutical compositions, kits, and articles of manufacture that include the compositions described herein.

[0259] In some aspects, provided herein are pharmaceutical compositions comprising at least four subspecies of DAS181 that can be separated by CEX-HPLC, the compositions comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via the C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid positions being relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via the C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein, the pharmaceutical compositions being formulated according to any of the formulations described in Section II.2 above. In some embodiments, the formulation is a dry powder microparticle formulation. In some embodiments, the formulation is a lyophilized cake formulation. In some embodiments, the formulation is reconstituted in an aqueous medium.

[0260] In some embodiments, the pharmaceutical composition comprises at least about 90% trehalose by dry weight. In some embodiments, the pharmaceutical composition comprises at least about 0.2% MgSO4 by dry weight. In some embodiments, the pharmaceutical composition comprises a) about 95-98% trehalose, b) about 0.2-0.4 MgSO4, c) about 0.4-0.6% sodium acetate, and d) about 0.1-0.3% acetic acid.

[0261] In some embodiments, provided herein is a pharmaceutical composition comprising: a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain; b) about 95-98% trehalose; c) about 0.2-0.4 MgSO4; d) about 0.4-0.6% sodium acetate; and d) about 0.1-0.3% acetic acid.

[0262] In some embodiments, the composition does not contain histidine or CaCl. In some embodiments, the pharmaceutical composition comprises about 97.5% trehalose, about 0.3% MgSO, about 0.5% sodium acetate, and about 0.2% acetic acid.

[0263] In some embodiments, the composition does not include histidine or CaCl2.

[0264] In some embodiments, the potency of the sialidase in the pharmaceutical composition is about 540-740 U / mg protein. In some embodiments, the pharmaceutical composition after reconstitution into a liquid formulation has an osmolality of about 270-330 mOsm / kg. In some embodiments, the pharmaceutical composition after reconstitution into a liquid formulation has a viscosity of about 1.27-1.39 cps. In some embodiments, the pharmaceutical composition after reconstitution into a liquid formulation has a pH of about 4.5 to about 6.5. In some embodiments, at least about 95% of the protein in the pharmaceutical composition is monomeric.

[0265] Pharmaceutical compositions may be prepared in the form of lyophilized formulations or aqueous solutions by mixing the DAS181 compositions described herein having the desired purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonicity agents (e.g., sodium chloride), stabilizers, metal complexes (e.g., Zn-protein complexes), chelating agents such as EDTA, and / or nonionic surfactants.

[0266] The formulation may include a carrier. The carrier is a polymer that is soluble in the circulatory system and physiologically acceptable, where physiologically acceptable means that a person skilled in the art would accept the carrier as part of a therapeutic regimen when injected into a patient. The carrier is preferably relatively stable in the circulatory system and has an acceptable plasma half-life for clearance. Such polymers include, but are not limited to, soybean lecithin, oleic acid, and sorbitan trioleate.

[0267] The formulation may also contain other agents useful for maintaining pH, stabilizing the solution, or regulating osmotic pressure, including, but not limited to, salts, such as sodium chloride or potassium chloride, and carbohydrates, such as glucose, galactose, or mannose.

[0268] In some embodiments, the pharmaceutical composition is contained in a single-use vial, e.g., a single-use sealed vial. In some embodiments, the pharmaceutical composition is contained in a multi-use vial. In some embodiments, the pharmaceutical composition is contained in a container in bulk. In some embodiments, the pharmaceutical composition is stored frozen.

[0269] In some embodiments, the systems provided herein can be stably stored under cryopreservation conditions, e.g., at -80°C, and can be thawed as needed or desired prior to administration. For example, the systems provided herein can be stored at a storage temperature, e.g., -20°C or -80°C, for at least or about several hours, i.e., 1, 2, 3, 4, or 5 hours, or several days, including at least or about several years, for example, but not limited to, 1, 2, 3 years or more, e.g., at least or about 1, 2, 3, 4, or 5 hours to at least or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 9 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours, or 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days, or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 months, or 1, 2, 3, 4, or 5 years or more. The systems provided herein can also be stably stored under refrigerated conditions, for example, at 4° C., and / or shipped on ice to the site of therapeutic administration. For example, the systems provided herein can be stored at 4° C. or on ice for at least or about several hours, for example, but not limited to, 1, 2, 3, 4, or 5 hours to at least or about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours or more prior to therapeutic administration.

[0270] The present application further provides kits and articles of manufacture for use in any of the embodiments of the treatment methods described herein. The kits and articles of manufacture may include any one of the formulations and pharmaceutical compositions described herein.

[0271] In some embodiments, a kit is provided that includes an immunoconjugate described herein and instructions for administering the immunoconjugate to an individual, in some embodiments, the kit further includes instructions for treating cancer.

[0272] In some embodiments, a kit is provided that includes any one of the immunoconjugates described herein and instructions for treating cancer. In some embodiments, the kit further includes an immunotherapeutic agent (e.g., a cell therapy or any one of the immunotherapies described herein). In some embodiments, the kit further includes one or more additional therapeutic agents for treating cancer. In some embodiments, the immunoconjugate and / or the one or more immunotherapeutic agents are included in a single composition (e.g., a composition including an immune checkpoint inhibitor and an immunoconjugate). In some embodiments, the immunoconjugate and, optionally, the one or more additional immunotherapeutic agents and / or additional therapeutic agents for treating cancer are included in separate compositions.

[0273] The kit of the present invention is contained in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), and the like. The kit may optionally provide additional components, such as buffers and instruction information. Thus, the present application also provides articles of manufacture that include vials (such as sealed vials), bottles, jars, flexible packages, and the like.

[0274] In some aspects, provided herein is a vial containing any one of the pharmaceutical compositions described herein. In some embodiments, the vial is for single use.

[0275] In some embodiments, provided herein is a nebulizer that contains liquid formulation, and the liquid formulation is reconstituted from any one of the pharmaceutical compositions of lyophilized cake described herein.The dosage form or administration by nebulizer generally contains a large amount of water in addition to the active ingredient.Small amounts of other ingredients, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, or buffers, and other stabilizers and solubilizers, can also be included.

[0276] The nasal formulation may be administered as a nasal drop, a spray, an aerosol, or by any other intranasal dosage form. Optionally, the delivery system may be a unit dose delivery system. The volume of the solution or suspension delivered per dose may be either about 5 to about 2000 microliters, about 10 to about 1000 microliters, or about 50 to about 500 microliters. The delivery systems for these various dosage forms may be dropper bottles, plastic squeeze units, atomizers, nebulizers, or aerosolized pharmaceuticals in unit dose or multi-dose packages.

[0277] The liquid formulations disclosed herein may be modified to include: (1) other acids and bases to adjust pH, (2) other isotonicity agents, such as sorbitol, glycerin, dextrose, (3) other antimicrobial preservatives, such as other parahydroxybenzoates, sorbates, benzoates, propionates, chlorobutanol, phenylethyl alcohol, benzalkonium chloride, and mercurials, (4) other viscosity agents, such as sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol, and other gums, (5) suitable absorption enhancers, (6) stabilizers, such as antioxidants such as bisulfites and ascorbates, metal chelators, such as sodium edetate, and drug solubility enhancers, such as polyethylene glycol, and (7) other agents, such as amino acids.

[0278] In some embodiments, provided herein are liquid pharmaceutical compositions with various dosage levels, for example, dosage levels of about 0.01 mg to about 100 mg of DAS181 active agent (or another polypeptide having sialidase activity). Examples of such dosage levels include dosages of about 0.05 mg, 0.06 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 50 mg, or 100 mg per day. The aforementioned dosages can be administered one or more times per day for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. Higher or lower doses can also be administered. Typically, dosages can be about 1 ng / kg to about 10 mg / kg, about 10 ng / kg to about 1 mg / kg, and about 100 ng / kg to about 100 micrograms / kg. In various examples described herein, mice were treated with various doses of the compositions described herein, including doses of 0.0008 mg / kg, 0.004 mg / kg, 0.02 mg / kg, 0.06 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.6 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, and 5.0 mg / kg.

[0279] Also provided herein is an article of manufacture comprising a microparticle formulation as described in Section II.2.A, comprising a sialidase or sialidase fusion protein in an amount of about 60% to about 75% w / w, about 95-98% w / w trehalose, about 0.2-0.4% w / w MgSO4, about 0.4-0.6% w / w sodium acetate, about 0.1-0.3% w / w acetic acid, packaging material for the formulation, and a label indicating that the composition is for a therapeutic indication. In one embodiment, the therapeutic indication is influenza. In another embodiment, the therapeutic indication is asthma or COPD. In yet another embodiment, the therapeutic indication is selected from among parainfluenza, RSV, sinusitis, otitis, laryngitis, bronchitis, pneumonia, bronchiectasis, vasculitis, mucus plugs, Wegener's granulomatosis, and cystic fibrosis (CF).

[0280] In some embodiments, the scavenger is histidine, while in other embodiments, the scavenger is a combination of histidine and trehalose, hi still other embodiments, the counterion is selected from among citrate / citrate, magnesium sulfate, potassium or calcium sulfate, phosphate, pivalate, rubidium, bromine, perchlorate, itaconate, and any salt, acid, or base form thereof.

[0281] The packaging material can be an HPMC capsule, and in further embodiments, the HPMC capsule is transparent. In other embodiments, the packaging material can be a gel capsule or a pullulan polysaccharide capsule. In still other embodiments, the product further comprises a secondary packaging material, and in some embodiments, the secondary packaging material is a foil laminate, and in certain embodiments, the foil laminate is a cold foil aluminum laminate blister pack. In some embodiments, the scavenging agent is an amine, and in further embodiments, the amine is selected from lysine, histidine, glycine, arginine, glutamine, glutamic acid, cysteine, alanine, tyrosine, tryptophan, aminoguanidine, cysteamine, serine, carnosine, hydralazine, and poly(1-lysine).

[0282] In some embodiments, the clearing agent is histidine or a combination of histidine and trehalose. In some embodiments, the article of manufacture includes a package insert containing instructions for reconstituting the lyophilized cake formulation described herein into a liquid formulation for administration via a nebulizer (e.g., a vibrating mesh nebulizer). In some embodiments, the liquid formulation includes 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0283] The articles of manufacture provided herein can also include an inhaler for pulmonary administration of the composition. In certain embodiments, the inhaler is a dry powder inhaler, a metered dose inhaler, or an electrostatic delivery device.

[0284] Also provided herein is an article of manufacture comprising the lyophilized cake formulation described in Section II.2.A, comprising a sialidase or sialidase fusion protein in an amount of about 1% w / w to about 2% w / w, a counterion and a scavenging agent that is a primary amine in an amount of about 8% w / w to about 11% w / w, packaging material for the formulation, and a label indicating that the composition is for a therapeutic indication. In some embodiments, the packaging material is a sealed glass vial. In some embodiments, the article of manufacture comprises a label providing instructions for reconstituting the lyophilized cake formulation in an aqueous medium. In some embodiments, the target inhalation volume is 3.5 mL and the reconstitution volume of a single glass vial is 3.8 mL. In one embodiment, the therapeutic indication is influenza. In other embodiments, the therapeutic indication is asthma or COPD. In still other embodiments, the therapeutic indication is selected from among parainfluenza, RSV, sinusitis, otitis, laryngitis, bronchitis, pneumonia, bronchiectasis, vasculitis, mucus plug, Wegener's granulomatosis, and cystic fibrosis (CF). In some embodiments, the article of manufacture further comprises a secondary packaging material. In some embodiments, the secondary packaging material is a container comprising a plurality of sealed glass vials.

[0285] The articles of manufacture provided herein can also include a nebulizer for pulmonary administration of the composition.

[0286] V. Treatment method In some aspects, provided herein are methods of treating a disease or condition in an individual, the methods comprising administering to the individual an effective amount of any of the pharmaceutical compositions described herein.

[0287] In some aspects, provided herein are methods of treating a disease or condition in an individual, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising at least four subspecies of DAS181 that can be separated by CEX-HPLC, the composition comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid position relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, the truncated cationic domain comprising amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein. In some embodiments, the pharmaceutical composition is formulated according to any of the formulations described in Section II.2 above. In some embodiments, the formulation is a dry powder microparticle formulation. In some embodiments, the formulation is a lyophilized cake formulation. In some embodiments, the formulation is reconstituted in an aqueous medium.

[0288] In some embodiments, provided herein are methods of treating a disease or condition in an individual, the methods comprising administering to the individual an effective amount of a pharmaceutical composition comprising, by dry weight, a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain, b) about 95-98% w / w trehalose, c) about 0.2-0.4% w / w MgSO4, d) about 0.4-0.6% w / w sodium acetate, and e) about 0.1-0.3% w / w acetic acid. In some embodiments, the pharmaceutical composition comprises, by dry weight, about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid. In some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition. In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid. In some embodiments, the fusion protein is DAS181, the sequence of which is shown in SEQ ID NO:1 (with an amino-terminal methionine) and SEQ ID NO:2 (without an amino-terminal methionine).In some embodiments, the fusion protein is A) a polypeptide comprising (or consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO:1, B) a polypeptide comprising (or consisting of or consisting essentially of) the amino acid sequence of SEQ ID NO:2, or C) a mixture of a polypeptide comprising (or consisting of or consisting essentially of) SEQ ID NO:1 and a polypeptide comprising (or consisting of or consisting essentially of) SEQ ID NO:2.

[0289] In some embodiments, provided herein are methods of treating a disease or condition in an individual, the method comprising administering to the individual: i) a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, the deaminated form of the first fusion protein comprising a deaminated N-residue compared to the first fusion protein, the amino acid positions being relative to SEQ ID NO:1; c) a truncated form of the sialidase domain fused via its C-terminus to a cationic domain, the cationic domain comprising amino acids 395-415 of SEQ ID NO:1; ii) about 95-98% w / w trehalose, iii) about 0.2-0.4% w / w MgSO4, iv) about 0.4-0.6% w / w sodium acetate, and v) about 0.1-0.3% w / w acetic acid, by dry weight. In some embodiments, the pharmaceutical composition comprises about 97.5% w / w trehalose, about 0.3% w / w MgSO4, about 0.5% w / w sodium acetate, and about 0.2% w / w acetic acid, by dry weight. In some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition. In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid.In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0290] In some embodiments, the disease or condition is a viral infection or is associated with a viral infection. In some embodiments, the viral infection is a coronavirus infection (e.g., SARS-CoV, MERS-CoV, or SARS-CoV-2), influenza infection, lower respiratory tract parainfluenza infection, BK polyomavirus, Merkel cell polyomavirus (MCPyV), or human enterovirus D68 (EV-D68) infection. DAS181 has been shown to have potent antiviral activity against all types of influenza, including pandemic strains such as H1N1 and avian influenza. DAS181 is also active against drug-resistant IFV, including strains resistant to Tamiflu (oseltamivir) and Relenza (zanamivir). Asian avian influenza A (H7N9) virus and two additional respiratory viruses that cause severe disease and rely on SA to infect cells, metapneumovirus and enterovirus 68, are also inhibited by DAS181.

[0291] In some embodiments, provided herein is a method for the prevention, prophylaxis, or treatment of diseases of the airways, including influenza, parainfluenza, RSV, sinusitis, otitis, laryngitis, bronchitis, pneumonia, allergic and non-allergic asthma, COPD, bronchiectasis, vasculitis, mucus plugging, Wegener's granulomatosis, and cystic fibrosis (CF), comprising administering to a patient in need thereof any of the pharmaceutical compositions described herein.

[0292] Therapeutic and diagnostic applications of the pharmaceutical compositions disclosed herein can include drug delivery, vaccination, gene therapy, and in vivo tissue or tumor imaging. Routes of administration can include oral or parenteral administration, mucosal administration, ophthalmic administration, intravenous, subcutaneous, intraarticular, or intramuscular injection, inhalation administration, and topical administration.

[0293] In some embodiments, provided herein is a method for treating a parainfluenza virus (PIV) or influenza virus (IFV) infection in a patient, comprising administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein to the airway of the patient. Also described herein is a method for treating a subject at risk of PIV or IFV infection, comprising administering any of the pharmaceutical compositions described herein to the airway of the subject. Methods for treating or preventing PIV or IFV infection, comprising administering a protein having sialidase activity, are described in US20150132274, the contents of which are incorporated herein by reference in their entirety. In various cases, i.e., the patient is an immunocompromised patient, the patient suffers from a primary immunodeficiency, the patient suffers from a secondary immunodeficiency, the patient is undergoing or has undergone immunosuppressive therapy, the patient is being treated or has been treated with a chemotherapy agent, the patient is a transplant patient, the composition further comprises one or more additional compounds, the administration is by using a dry powder inhaler, the administration is by using a nasal spray, the administration is by using a nebulizer, the administration is by using an endotracheal tube (ET tube) and a dry powder inhaler. In some cases, the patient has insufficient lung function to effectively use a dry powder inhaler or cannot use a dry powder inhaler at all, for example, the patient is on a ventilator. In some cases, the patient is an immunocompromised patient infected with PIV and is treated with a liquid formulation (e.g., using a nebulizer) or with a dry formulation (e.g., using a dry powder inhaler).In some embodiments, the pharmaceutical composition comprises a mixture of DAS181 subspecies that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain.

[0294] In some aspects, provided herein is a method of treating an infection with BK polyomavirus or a disorder associated with BK polyomavirus in a patient, comprising administering to the patient a therapeutically effective amount of an agent having sialidase activity contained in any of the pharmaceutical compositions disclosed herein. A method of treating an infection with BK polyomavirus or a disorder associated with BK polyomavirus, comprising administering a protein having sialidase activity, is described in US10300116, the contents of which are incorporated herein by reference in their entirety. In various embodiments, the patient is immunocompromised, the patient has undergone hematopoietic stem cell transplantation or is preparing for hematopoietic stem cell transplantation, the disorder is BKV nephropathy, the disorder is nephritis, the disorder is hemorrhagic cystitis, the disorder is ureteral stenosis, the patient has undergone solid organ transplantation or is undergoing treatment in preparation for solid organ transplantation, and the disorder is lupus. In some embodiments, the pharmaceutical composition comprises a mixture of subspecies of DAS181 that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some embodiments, the pharmaceutical composition comprises, by dry weight, about 97.5% trehalose, about 0.3% MgSO4, about 0.5% sodium acetate, and about 0.2% acetic acid, hi some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition.In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0295] In some embodiments, provided herein is a method of treating an infection with Merkel cell polyomavirus (MCPyV) or an MCPyV-associated disorder, comprising administering to the skin of the patient a therapeutically effective amount of an agent having sialidase activity contained in any of the pharmaceutical compositions disclosed herein. A method of treating an MCPyV infection or an MCPyV-associated disorder, comprising administering a protein having sialidase activity, is described in US20200222511, the contents of which are incorporated herein by reference in their entirety. In various embodiments, the patient is immunocompromised, the patient is infected with HIV, the patient suffers from chronic lymphocytic leukemia, the patient has undergone an organ transplant or is undergoing treatment in preparation for an organ transplant, the patient has undergone a liver, heart, bone marrow, or kidney transplant or is undergoing treatment in preparation for a liver, heart, bone marrow, or kidney transplant. In some embodiments, the pharmaceutical composition comprises a mixture of subspecies of DAS181 that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some embodiments, the pharmaceutical composition comprises, by dry weight, about 97.5% trehalose, about 0.3% MgSO4, about 0.5% sodium acetate, and about 0.2% acetic acid, hi some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition.In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0296] In some embodiments, provided herein is a method of treating an infection caused by MERS-CoV or a MERS-CoV-related disorder in a patient, comprising administering to the patient a therapeutically effective amount of an agent having sialidase activity comprised in any of the pharmaceutical compositions disclosed herein. A method of treating an infection caused by MERS-CoV or a MERS-CoV-related disorder in a patient comprising administering a protein having sialidase activity is described in US20200397871, the contents of which are incorporated herein by reference in their entirety. In various embodiments, the patient is immunocompromised, the patient is receiving immunosuppressive therapy, and / or the patient is over 70 years of age. In some embodiments, the pharmaceutical composition comprises a mixture of subspecies of DAS181 that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some embodiments, the pharmaceutical composition comprises, by dry weight, about 97.5% trehalose, about 0.3% MgSO4, about 0.5% sodium acetate, and about 0.2% acetic acid, hi some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition.In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0297] In some embodiments, provided herein is a method of treating an infection by human enterovirus D68 (EV-D68) (species: Human enterovirus D, genus: Enterovirus, family: Picornaviridae) or an EV-D68-associated disorder in a patient, comprising administering to the patient a therapeutically effective amount of an agent having sialidase activity comprised in any of the pharmaceutical compositions disclosed herein. A method of treating an EV-D68 infection or an EV-D68-associated disorder in a patient, comprising administering a protein having sialidase activity, is described in US10328128, the contents of which are incorporated herein by reference in their entirety. In various embodiments, the patient is immunocompromised, the patient is receiving immunosuppressive therapy, the patient is over 70 years old, the patient is under 18 years old, or the patient is under 10 years old. In some embodiments, the pharmaceutical composition comprises a mixture of subspecies of DAS181 that can be separated by CEX-HPLC, including: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, wherein the cationic domain comprises amino acids 395-415 of SEQ ID NO:1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N-residue compared to the first fusion protein, and wherein the amino acid position is relative to SEQ ID NO:1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-406 of SEQ ID NO:1; and d) a fourth subspecies comprising a dimerized form of the first fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain. In some embodiments, the pharmaceutical composition comprises, by dry weight, about 97.5% trehalose, about 0.3% MgSO4, about 0.5% sodium acetate, and about 0.2% acetic acid, hi some embodiments, the pharmaceutical composition is a liquid composition reconstituted from a lyophilized composition.In some embodiments, the liquid formulation comprises 1-2 mg / mL DAS181 and DAS181 variant subspecies, 10-100 mg / mL trehalose, 0.1-0.5 mg / mL MgSO4, 0.2-0.7 mg / mL sodium acetate, and 0.1-0.3 mg / mL acetic acid. In some embodiments, the liquid formulation comprises about 1.3 mg / mL DAS181 and DAS181 variant subspecies, about 94 mg / mL trehalose, about 0.3 mg / mL MgSO4, about 0.5 mg / mL sodium acetate, and about 0.2 mg / mL acetic acid. In some embodiments, the liquid formulation comprises 1.3 mg / mL DAS181 and DAS181 variant subspecies, 94 mg / mL trehalose, 0.3 mg / mL MgSO4, 0.5 mg / mL sodium acetate, and 0.2 mg / mL acetic acid.

[0298] In some embodiments, the method comprises administering the pharmaceutical composition by inhalation.When administering by inhalation, the pharmaceutical composition can be delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.Such methods include those described in U.S. Patent No. 6,468,798.In some embodiments, the drug(s) is administered to the subject by using a nebulizer, a vaporizer, a nasal spray, a pressurized metered-dose inhaler, or a breath-actuated pressurized metered-dose inhaler.

[0299] In some embodiments, the device is a small hard bottle with a metered dose sprayer attached. A metered dose can be delivered by drawing the composition into a chamber with a specified volume. The chamber has an aperture with a size for aerosolizing the aerosol formulation by forming a spray when the liquid in the chamber is compressed. The chamber is compressed to administer the composition. In some devices, the chamber is a piston mechanism. Such devices are commercially available.

[0300] Alternatively, a squeeze bottle can be used that has an aperture or opening sized to aerosolize the aerosol formulation by forming a spray when squeezed. The opening is usually at the top of the bottle, which is generally tapered to partially fit into the nasal cavity for efficient administration of the aerosol formulation. Preferably, the nasal inhaler can provide a metered amount of the aerosol formulation to administer a measured dose of the therapeutic agent.

[0301] In some embodiments, the method comprises administering the pharmaceutical composition using a nebulizer, for example, a vibrating mesh nebulizer. In some embodiments, the total protein (DAS181, or DAS181 and DAS181 variant subspecies) added to the nebulizer is 0.65-4.5 mg / day. In some embodiments, the dose of total protein (DAS181, or DAS181 and DAS181 variant subspecies) added to the nebulizer is 4.5 mg / day for adult patients (e.g., patients 18 years of age or older). In some embodiments, the dose of total protein (DAS181, or DAS181 and DAS181 variant subspecies) added to the nebulizer is 2.5 mg / day for pediatric patients. In some embodiments, the pediatric patient is a patient weighing less than 40 kg.

[0302] While certain embodiments have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the present disclosure described herein may be used in implementing the present disclosure. It is intended that the following claims define the scope of the present disclosure, and that the methods and structures within these claims and their equivalents are covered thereby. EXAMPLES

[0303] Example 1. Characterization of compositions containing DAS181 variants by CEX-HPLC This example describes the characterization of compositions containing DAS181 subspecies by CEX-HPLC.

[0304] A standard lyophilization manufacturing process was developed to produce a sterile lyophilized cake containing DAS181 variant in a glass vial configuration. Lyophilization parameters including freezing and heating gradients, primary and secondary drying temperatures and times, and vacuum settings were evaluated and selected based on the appearance of the final product cake.

[0305] The composition was subjected to cation exchange chromatography (CEX-HPLC) using nonporous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acids and eluted with a gradient of up to 200 mM CaCl in acetate buffer at a flow rate of 1 mL / min. The results of this CEX-HPLC are shown in FIG.

[0306] As shown in Figure 1, the composition is composed of at least four subspecies, including the DAS181 subspecies that can be separated by CEX-HPLC. The first subspecies eluted at about 84 mM CaCl2. The second subspecies eluted at about 72 mM CaCl2. The third subspecies eluted at about 57.5 mM CaCl2. The fourth subspecies eluted at about 200 mM CaCl2. The composition further contained a fifth subspecies, which was not isolated for further analysis. The fifth subspecies eluted at about 100 mM CaCl2.

[0307] Example 2. Characterization of DAS181 variants This example describes the intact mass analysis of the first, second, third, and fourth DAS181 variants identified by CEX-HPLC.

[0308] Approximately 5 μg of sample was loaded onto the analytical SEC-MS column. MS data were collected using an Agilent 6550 iFunnel QTOF. MS analysis was performed using Agilent Bioconfirm software.

[0309] The SEC-MS method was performed using the following parameters: isocratic, mobile phase: 30% w / v acetonitrile, LC / MS water with 0.1% w / v formic acid, column: Waters BEH SEC column, 200A, 1.7um, 4.6x300mm.

[0310] Deconvolution parameters for intact mass analysis were as follows: deconvolution algorithm: maximum entropy, mass range: 30 kDa–150 kDa, mass step: 0.5 Da, M / Z range: 650–4000 Da, baseline factor: 7.0, adduct: proton, isotope width: automatic.

[0311] First variant The results of the intact mass analysis of the first species are shown in FIG. 2 and Table E1 below. [Table 2]

[0312] Second variant The results of the intact mass analysis of the first species are shown in FIG. 3 and Table E2 below. [Table 3]

[0313] Third variant The results of the intact mass analysis of the first species are shown in FIG. 4 and Table E3 below. [Table 4]

[0314] Fourth variant The results of the intact mass analysis of the first species are shown in FIG. 5 and Table E4 below. [Table 5]

[0315] The fourth subspecies was analyzed by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) to identify the oligomeric state of the protein contained in the fourth subspecies. The results of the SEC-HPLC experiment are shown in Figure 6, and the quantification of the peaks is shown in Table E6 below. Based on SEC, the fourth subspecies contained about 35% by weight dimer and about 64% by weight monomer. [Table 6]

[0316] Size variant distribution by SDS-PAGE As shown in Figure 7, the identity of the DAS181 variants was confirmed by SDS-PAGE with Coomassie blue staining and comparison with the reference standard. The location of the major bands of all drug product lots detected by Coomassie staining matched the major band of the DAS181 reference standard. All minor bands observed in reduced silver-stained SDS-PAGE were DAS181-related, and those differences were due to lot-to-lot variation of the drug substance. The minor differences observed in the silver-stained SDS-PAGE gels are unlikely to affect the safety and efficacy of the product.

[0317] Example 3. Activity of DAS181 variants This example demonstrates the sialidase activity of the isolated first, second, third, and fourth DAS181 variants. Sialidase activity was measured using the fluorogenic substrate 2'-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (MuNaNa), VWR (Biosynth) catalog number 101369-938 (M-5507). Sialidase (DAS181 variants) cleaves sialic acid from this substrate, and free sialic acid is detected in the assay. As shown in Table E7 below, all DAS181 variants retained sialidase activity. Results of two different purification lots measured in two independent experiments are shown. The control for sialidase activity measurement was the first fusion protein. [Table 7]

[0318] Example 4. Lyophilized cake formulation of DAS181 In this comparative study, a reconstituted dry lyophilized microparticle formulation (based on the formulation previously described in US 9,700,602) and the new lyophilized cake formulation disclosed herein were tested after storage at 25°C / 60%RH for up to 2 weeks. Initial (time 0), 24 hour, and 1 week samples were drawn and stored at -80°C until the 2 week sample was drawn. All samples were tested together. The most sensitive stability indicating methods for DAS181 were used in this comparative evaluation, including purity by CEX-HPLC and % monomer by SEC-HPLC. Deamidation is the major degradation pathway of DAS181 in solution. The second variant is mainly the deamidated product of peak A and retains full potency. The percentage of the second variant in the lyophilized cake formulation is 16.8% at time 0 and 28.0% at 2 weeks. The percentage of the second subspecies in the dried microparticle formulation is 10.8% at time 0 and 28.8% at 2 weeks. The percentage increase in peak C in the reconstituted DAS181-SF1 lot was 11.2% at 2 weeks, while the percentage increase in the second subspecies in the reconstituted dried microparticle formulation was 18.0% at 2 weeks. Thus, the reconstituted lyophilized cake formulation is more stable than the reconstituted dried microparticle formulation when stored at 25°C / 60% RH. Representative CEX-HPLC chromatograms of the reconstituted dried microparticle formulation and the reconstituted lyophilized cake formulation are shown in Figures 8A and 8B, respectively. Table E8 below shows the quantification of the peak areas at various time points. Table E9 shows the SEC-HPLC and activity measurements of the lyophilized cake formulations after storage at room temperature for 6-8 hours, 24 hours (± 1 hour), 48 hours (± 1 hour), 3 days, or 7 days. [Table 8] [Table 9]

[0319] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly indicated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0320] Example 5. Update of the lyophilization cycle for the lyophilized cake formulation of DAS181 This example describes an update of the lyophilization cycle of a DAS181 formulation to use an increased primary drying temperature and a reduced total lyophilization time from 103 hours to 63 hours (see, e.g., Table E9) compared to a previously described lyophilization cycle (see, e.g., US 9,700,602, the contents of which are incorporated herein by reference in their entirety). The resulting product was a white lyophilized cake that formed a clear, colorless solution after reconstitution with no visible residue or undissolved material. [Table 10] Exemplary Sequences SEQ ID NO:1 DAS181 MGDHPQATPAPPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKV GYSDPSYVVDHQTGTIFHFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAG GAVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSH SPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP SEQ ID NO:2 DAS181 No N-terminal Met GDHPQATPAPAPDASTELPASMSQAQHLAANTATDNYRIPAITTAPNGDLLISYDERPKDNGNGGSDAPNPNHIVQRRSTDGGKTWSAPTYIHQGTETGKKVG YSDPSYVVDHQTGTIFHFHVKSYDQGWGGSRGGTDPENRGIIQAEVSTSTDNGWTWTHRTITADITKDKPWTARFAASGQGIQIQHGPHAGRLVQQYTIRTAGG AVQAVSVYSDDHGKTWQAGTPIGTGMDENKVVELSDGSLMLNSRASDGSGFRKVAHSTDGGQTWSEPVSDKNLPDSVDNAQIIRAFPNAAPDDPRAKVLLLSH SPNPRPWSRDRGTISMSCDDGASWTTSKVFHEPFVGYTTIAVQSDGSIGLLSEDAHNGADYGGIWYRNFTMNWLGEQCGQKPAKRKKKGGKNGKNRRNRKKKNP

Claims

1. 1. A composition comprising at least four subspecies of DAS181 that can be separated by cation exchange high performance liquid chromatography (CEX-HPLC), the composition comprising: a) a first subspecies comprising a first fusion protein comprising a sialidase domain fused via its C-terminus to a cationic domain, said cationic domain comprising amino acids 395-415 of SEQ ID NO: 1; b) a second subspecies comprising a deaminated form of the first fusion protein, wherein the deaminated form of the first fusion protein comprises a deaminated N residue compared to the first fusion protein, and the amino acid positions are relative to SEQ ID NO: 1; c) a third subspecies comprising a second fusion protein comprising a sialidase domain fused via its C-terminus to a truncated cationic domain, wherein the truncated cationic domain comprises amino acids 395-397 of SEQ ID NO: 1; and d) A fourth variant comprising a dimerized form of said first fusion protein.

2. 2. The composition of claim 1, wherein the deaminated N residue in the second subspecies is N403 and / or the fourth subspecies comprises a methionine-oxidized form of the first fusion protein.

3. 10. The composition of claim 1, further comprising a fifth subspecies comprising a dehydrated form of the first fusion protein.

4. 10. The composition of claim 1, wherein the third subspecies further comprises an oxidized or deaminated form of the second fusion protein.

5. 2. The composition of claim 1, wherein the first subspecies further comprises a third fusion protein lacking an N-terminal M residue compared to the first fusion protein.

6. 6. The composition of claim 5, wherein the ratio of the first fusion protein to the third fusion protein in the first subspecies is about 2:

1.

7. 2. The composition of claim 1, wherein the sialidase domain comprises amino acids 1-394 of SEQ ID NO:

1.

8. 2. The composition of claim 1, wherein the first fusion protein comprises amino acids 1 to 415 of SEQ ID NO:

1.

9. 2. The composition of claim 1, wherein the second fusion protein comprises amino acids 1 to 406 of SEQ ID NO:

1.

10. 10. The composition of claim 1, wherein the weight percentage of the first subspecies of the total protein in the composition is about 50% to about 90%, the weight percentage of the second subspecies of the total protein in the composition is about 10% to about 30%, the weight percentage of the third subspecies of the total protein in the composition is about 1% to about 15%, or the weight percentage of the fourth subspecies of the total protein in the composition is about 0.1% to about 4%.

11. 4. The composition of claim 3, wherein the weight percentage of the fifth subspecies in the total protein in the composition is about 1% to about 10%.

12. The composition was subjected to cation exchange chromatography (CEX-HPLC) using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acids, and the beads were diluted with acetate buffer containing up to 200 mM CaCl ２ The first subspecies was eluted with a gradient containing about 80-85 mM CaCl ２ 10. The composition of claim 1, wherein the first subspecies elutes at about 70-75 mM CaCl 2 , the second subspecies elutes at about 70-75 mM CaCl 2 , the third subspecies elutes at about 57-58 mM CaCl 2 , or the fourth subspecies elutes at about 180-200 mM CaCl 2 .

13. The composition was subjected to cation exchange chromatography (CEX-HPLC) using non-porous 3 μm polystyrene divinylbenzene beads derivatized with carboxylic acids, and the beads were diluted with acetate buffer containing up to 200 mM CaCl ２ The fifth subspecies was found to be eluted with a gradient containing about 95-105 mM CaCl ２ The composition of claim 3, wherein the composition is eluted at 0.5 wt.

14. The composition of any one of claims 1 to 9, wherein the second fusion protein is capable of binding to the cell surface of an airway epithelium.

15. A pharmaceutical composition comprising the composition according to any one of claims 1 to 9.

16. 10. A method of releasing a composition according to any one of claims 1 to 9 for use in human medicine, said method comprising subjecting said composition to CEX-HPLC and determining the relative amounts of said first, second, third, fourth and optionally fifth subspecies separated by said CEX-HPLC.

17. 17. The method of claim 16, wherein the weight percentage of the first subspecies of the total protein in the composition is about 50% to about 90%, indicating that the composition is suitable for human medical use; the weight percentage of the second subspecies of the total protein in the composition is about 15% to about 30%, indicating that the composition is suitable for human medical use; the weight percentage of the third subspecies of the total protein in the composition is about 1% to about 15%, indicating that the composition is suitable for human medical use; or the weight percentage of the fourth subspecies of the total protein in the composition is about 0.1% to about 4%, indicating that the composition is suitable for human medical use.

18. The method described in claim 16, wherein, when the first subspecies is present, the weight percentage of the fifth subspecies of the total protein contained in the composition is about 1% to about 10%, indicating that the composition is suitable for human medical use.

19. 18. A pharmaceutical composition released for human medical use by the method of claim 17.

20. A method of making a pharmaceutical composition comprising the composition of any one of claims 1 to 9, comprising: a) introducing a nucleic acid encoding the protein of SEQ ID NO: 1 into a bacterial host cell; b) expressing in said bacterial host cell the protein encoded by said nucleic acid; c) purifying the protein by chromatography to obtain a purified protein composition; and d) assessing the suitability of said purified protein composition for human medical use. Including, The method, wherein said evaluating comprises subjecting said purified protein composition to CEX-HPLC and identifying the relative amounts of said first, second, third, fourth, and optionally fifth subspecies separated by said CEX-HPLC.

21. 21. The method of claim 20, further comprising formulating the purified protein composition to obtain a pharmaceutical composition.

22. 21. A pharmaceutical composition prepared according to the method of claim 20.

23. Furthermore, at least about 70% trehalose by dry weight; at least about 0.2% MgSO 4 by dry weight, or a) about 95-98% w / w trehalose, b) about 0.2-0.4% w / w MgSO 4 , c) about 0.4-0.6% w / w sodium acetate, and d) about 0.1-0.3% w / w acetic acid.

16. The pharmaceutical composition of claim 15, comprising:

24. a) a fusion protein comprising a sialidase domain fused at its C-terminus to a cationic domain, b) about 95-98% w / w trehalose, c) about 0.2-0.4% w / w MgSO ４ d) about 0.4-0.6% w / w sodium acetate; and e) about 0.1-0.3% w / w acetic acid.

25. 16. The pharmaceutical composition of claim 15, formulated as a lyophilized formulation.

26. 16. A liquid formulation reconstituted from the pharmaceutical composition of claim 15.

27. 16. A vial containing any one of the pharmaceutical compositions of claim 15.

28. 16. A nebulizer containing a liquid formulation, wherein the liquid formulation is reconstituted from the pharmaceutical composition of claim 15.

29. A commercial batch comprising the pharmaceutical composition of claim 15.

30. A pharmaceutical composition according to claim 15, or a liquid formulation reconstituted from said pharmaceutical composition, for use in the treatment of a disease.

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