EP4319818A1 - Formulations of l-asparaginase - Google Patents

Formulations of l-asparaginase

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Publication number
EP4319818A1
EP4319818A1 EP22724215.3A EP22724215A EP4319818A1 EP 4319818 A1 EP4319818 A1 EP 4319818A1 EP 22724215 A EP22724215 A EP 22724215A EP 4319818 A1 EP4319818 A1 EP 4319818A1
Authority
EP
European Patent Office
Prior art keywords
formulation
asparaginase
administered
present disclosure
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22724215.3A
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German (de)
English (en)
French (fr)
Inventor
Sekhar Kanapuram
Haripada MAITY
Guifeng Jiang
Roja NARWAL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jazz Pharmaceuticals Ireland Ltd
Original Assignee
Jazz Pharmaceuticals Ireland Ltd
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Filing date
Publication date
Application filed by Jazz Pharmaceuticals Ireland Ltd filed Critical Jazz Pharmaceuticals Ireland Ltd
Publication of EP4319818A1 publication Critical patent/EP4319818A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)

Definitions

  • L- asparaginases Proteins with L-asparagine aminohydrolase activity, commonly known as L- asparaginases, have successfully been used for the treatment of various diseases that are potentially fatal, including cancers such as Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LBL), for which children constitute a large proportion of patients stricken with these diseases.
  • ALL Acute Lymphoblastic Leukemia
  • LBL Lymphoblastic Lymphoma
  • L-asparaginases of bacterial origin have a high immunogenic and antigenic potential.
  • E. coli derived L-asparaginase and pegaspargase are E. coli derived L-asparaginase and pegaspargase. These products can provoke adverse hypersensitivity reactions including allergic reaction, silent inactivation, and anaphylactic shock in patients.
  • Erwinaze® has been plagued by supply issues for years: reportedly, it can take 9 months to prepare (See, e.g., “Saving Ava: When a cancer drug shortage imperiled a toddler, her mom got to work”, Stat News, Karen Weintraub (Oct. 31, 2016); Asparaginase Erwinia Chrysanthemi Drug Shortage, Drugs.com). Even today the issues persist and Erwinase® shortages are common.
  • coli-derived asparaginase would address a significant medical need (as a component of a multi-agent chemotherapeutic regimen) for patients with ALL/Lymphoblastic Lymphoma (LBL), by helping to ensure availability of an asparaginase for patients who have developed hypersensitivity to E.coli-derived asparaginase.
  • LBL ALL/Lymphoblastic Lymphoma
  • Erwinase® is supplied as a sterile, lyophilized, white powder in vials. Each vial contains 10,000 International Units of asparaginase Erwinia chrysanthemi, and the following inactive ingredients: glucose monohydrate (5.0 mg), sodium chloride (0.5 mg).
  • non-lyophilized formulations removes the need for rehydration before administration to a subject, e.g., a human.
  • Oncaspar® an alternative, non- lyophilized treatment option, is supplied as a clear, colorless, preservative-free, isotonic sterile solution in phosphate-buffered saline, pH 7.3.
  • Each milliliter contains 750 ⁇ 150 International Units of pegaspargase, dibasic sodium phosphate, USP (5.58 mg), monobasic sodium phosphate, USP, (1.20 mg) and sodium chloride, USP (8.50 mg) in water for injection, USP.
  • ASPARLAS® another non-lyophilized alternative, is supplied as a clear, colorless, preservative-free, isotonic sterile solution in phosphate-buffered saline, pH 7.3, that requires dilution prior to intravenous infusion.
  • Each vial of ASPARLAS® contains 3,750 units in 5 mL of solution.
  • Each milliliter contains 750 units of calaspargase pegol-mknl; dibasic sodium phosphate, USP (5.58 mg); monobasic sodium phosphate, USP (1.20 mg); and sodium chloride, USP (8.50 mg) in water for injection, USP.
  • non-lyophilized formulations such as Oncaspar® and ASPARLAS®
  • stability for example, the existing formulations must be used within 48 hours if stored at room temperature.
  • such formulations may find use in the treatment of one or more diseases, disorders, or conditions treatable by asparagine depletion, including, for example, cancers such as Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LBL), including relapsed ALL and relapsed LBL.
  • ALL Acute Lymphoblastic Leukemia
  • LBL Lymphoblastic Lymphoma
  • aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
  • the one or more stabilizers comprise one or more disaccharides, one or more sorbitols, one or more amino acids, or any combination thereof.
  • the one or more disaccharides comprise trehalose, sucrose, or any combination thereof.
  • the one or more disaccharides comprise trehalose.
  • the one or more buffers are substantially free of amino acid.
  • the one or more buffers comprise a phosphate buffer, an acetate buffer, or any combination thereof.
  • the one or more buffers comprise a phosphate buffer.
  • the one or more buffers comprise sodium phosphate.
  • the sodium phosphate is sodium phosphate dibasic anhydrous, sodium phosphate monobasic monohydrate, or a combination thereof.
  • the formulation further comprises sodium chloride.
  • the formulation further comprises one or more excipients.
  • the one or more excipients comprise polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof.
  • the formulation has a pH of between about 4.0 and about 8.5.
  • the L-asparaginase is present at a concentration of about 20 mg/mL. In some embodiments, the L-asparaginase is non-PEGylated and non-PASylated.
  • the formulation comprises less than about 0.6% low- molecular-weight (LMW) species after storage at 40 °C for two months. In some embodiments, the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months.
  • LMW low- molecular-weight
  • HMW high-molecular-weight
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • provided herein is a method of treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject any of the formulations described herein.
  • a unit dosage form comprising: (i) any of the formulations described herein; and (ii) one or more pharmaceutically acceptable excipients.
  • a kit comprising: (i) any of the formulations described herein; and (ii) instructions for treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof.
  • Subject refers to mammals, and includes humans and non-human mammals. Examples of individuals include, but are not limited to mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cows, and humans. In some embodiments, subject refers to a human.
  • the articles “a” and “an” as used herein and in the appended claims are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise.
  • a buffer means one buffer or more than one buffer.
  • a parameter or value includes and describes that parameter or value per se.
  • “about X” includes and describes X per se.
  • the term “about” modifying, for example, the dimensions, volumes, quantity of an ingredient in a composition, concentrations, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations.
  • the term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities.
  • the term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent or less of the stated reference value.
  • the terms “treatable by depletion of asparagine” and “treatable by asparagine depletion” refers to a disease, condition, or disorder wherein the cells involved in or responsible for the disease, condition, or disorder either lack or have a reduced ability to synthesize L-asparagine. Depletion or deprivation of L-asparagine can be partial or substantially complete (e.g., to levels that are undetectable using methods and apparatus that arc known in the art). [0020]
  • the term “comprising the sequence of SEQ ID NO: 1” means that the amino-acid sequence of the protein may not be strictly limited to SEQ ID NO: I but may contain additional amino acids.
  • LMW species refers to species that have a molecular weight that is lower than that of L-asparaginase.
  • the LMW species have a molecular weight that is less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the molecular weight of L- asparaginase.
  • LMW species are formed by the degradation or decomposition of L-asparaginase, e.g., through hydrolysis of one or more covalent bonds in the L-asparaginase.
  • “High-molecular-weight species” or “HMW species”, as used herein, refers to species that have a molecular weight that is greater than that of L-asparaginase.
  • the HMW species have a molecular weight that is greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%, greater than about 300%, greater than about 350%, greater than about 400%, greater than about 450%, or greater than about 500% of the molecular weight of L-asparaginase.
  • Tehalose refers to a disaccharide having the following structure: .
  • Sucrose refers to a disaccharide having the following structure: .
  • Polysorbate 80 or “PS-80” refers to an excipient having the following structure: .
  • PEGylated is used to describe a structure or moiety to which PEG (polyethylene glycol) is conjugated or attached. Any suitable number of PEG units may be attached in any suitable way (e.g., through one or more covalent bonds) and at any suitable location of the structure or moiety in question.
  • a “PEGylated L- asparaginase” indicates an L-asparaginase to which one or more PEG units have been conjugated or attached.
  • a “non-PEGylated” moiety is one to which PEG has not been conjugated or attached.
  • PASylation is used to describe a structure or moiety to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, is conjugated attached. Any suitable number of such polypeptide units may be attached in any suitable way (e.g., through one or more covalent bonds) and at any suitable location of the structure or moiety in question.
  • a “PASylated L- asparaginase” indicates an L-asparaginase to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, have been conjugated or attached.
  • a “non-PASylated” moiety is one to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, has not been conjugated or attached.
  • “SEQ ID NO: 1” is as follows: ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQF SNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSD KPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYIT KTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYG YQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTG NGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHT
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
  • the L-asparaginase formulation comprises one or more stabilizers.
  • the one or more stabilizers comprise one or more disaccharides, one or more sorbitols, one or more amino acids, or any combination thereof.
  • the L-asparaginase formulation comprises one or more disaccharides. In some embodiments, the one or more disaccharides comprise trehalose, sucrose, or any combination thereof. In some embodiments, the L-asparaginase formulation comprises one or more buffers. In some embodiments, the one or more buffers, wherein the one or more buffers are substantially free of amino acid. In some embodiments, the L-asparaginase formulation comprises one or more stabilizers and one or more buffers. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low- molecular-weight (LMW) species after storage at 40 °C for two months.
  • LMW low- molecular-weight
  • the L-asparaginase formulation comprises less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months. In some embodiments, the L- asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species and less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • HMW high-molecular-weight
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • a L-asparaginase in accordance with the disclosure provided herein is an recombinant L-asparaginase.
  • a L-asparaginase in accordance with the invention described herein is an enzyme with L-asparagine aminohydrolase activity.
  • Such a L-asparaginase’s enzymatic activity may include not only deamidation of asparagine to aspartic acid and ammonia, but also deamidation of glutamine to glutamic acid and ammonia.
  • a L-asparaginase as disclosed herein is active as a multimer.
  • the L-asparaginase is an active enzyme as a tetramer.
  • a tetramer is composed of four subunits (also known as monomers).
  • a L-asparaginase is a tetramer consisting of four identical 35 kD subunits.
  • the L-asparaginase is a non-disulfide bonded tetrameric therapeutic protein.
  • each of the subunits or monomers of a multimeric L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1.
  • each of the subunits or monomers of a tetrameric Lasparaginase comprises the amino acid sequence of SEQ ID NO: 1.
  • the L-asparaginase is from Erwinia chrysanthemi NCPPB 1066 (Genbank Accession No. CAA32884, incorporated herein by reference in its entirety), either with or without signal peptides and/or leader sequences.
  • the L-asparaginase is composed of multiple subunits, for example, four subunits or monomers (tetramer).
  • a corresponding modified protein may then, e.g., consist of 1 to 20 (or more) peptides conjugated to each of the monomers of that tetramer.
  • the L-asparaginase comprises a monomer and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 (or more) peptides conjugated to each of the L-asparaginase monomers.
  • the L- asparaginase is a multimer comprising multiple subunits or monomers, such as a tetramer, and each of the monomers in that tetramer is conjugated to 1 peptide, resulting in a tetramer comprising 4 conjugated peptides, one for each monomer.
  • the L- asparaginase is a tetramer comprising 1-4 peptides conjugated to each of the monomers.
  • the L-asparaginase is a tetramer comprising 4-20 peptides conjugated to each of the L- monomers.
  • the Lasparaginase is a tetramer comprising 6-18 peptides conjugated to each of the monomers.
  • the L-asparaginase is a tetramer comprising 6-18 peptides conjugated to each of the monomers.
  • the L-asparaginase is a tetramer comprising 10-15 peptides conjugated to each of the monomers.
  • the invention relates to a modified protein having a L- asparaginase and multiple chemically attached peptide sequences.
  • the length of the peptide sequences are from about 10 to about 100, from about 15 to about 60 or from about 20 to about 40.
  • fragments of L-asparaginase may be of use in the presently described invention.
  • the term “a fragment of L-asparaginase” (e.g., a fragment of the L-asparaginase of SEQ ID NO: 1) means that the sequence of the L-asparaginase may include fewer amino-acids than in the L-asparaginases exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1) but still enough amino-acids to confer L-aminohydrolase activity.
  • a “fragment of L-asparaginase” is a fragment that is/consists of at least about 150 or 200 contiguous amino acids of one of the L- asparaginases exemplified herein (e.g. the L-asparaginase of SEQ ID NO: 1) (for example, about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, or 326 contiguous amino acids) and/or wherein said fragment has up to 50 amino acids deleted from the N-terminus of said L-asparaginases exemplified herein (e.g.
  • the L-asparaginase of SEQ ID NO: 1) e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50
  • has up to up to 75 or 100 amino acids deleted from the C- terminus of said L-asparaginases exemplified herein e.g., the Lasparaginase of SEQ ID NO: 1
  • the Lasparaginase of SEQ ID NO: 1 e.g.
  • L-asparaginases up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95 or 100
  • L-asparaginases exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1), wherein the total number of amino acids deleted can be up to 125 or 150 amino acids.
  • a Nessler assay is used for the determination of L-asparaginase activity according to a method described by Mashburn and Wriston (Mashburn, L., and Wriston, J. (1963) “Tumor Inhibitory Effect ofLAsparaginase,” Biochem Biophys Res Commun 12, 50, incorporated herein by reference in its entirety).
  • Mashburn and Wriston a method described by Mashburn and Wriston
  • a polypeptide can be modified by substitution, insertion, deletion and/or addition of one or more amino-acids while retaining its enzymatic activity.
  • the term "one or more amino acids” in this context can refer to one, two, three, four, five, six, seven, eight, nine, ten or more amino acids.
  • substitutions may be defined as exchanges within one of the following groups: Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; Polar, positively charged residues: His, Arg, Lys; Large aliphatic, non-polar residues: Met, Leu, Ile, Val, Cys; Large aromatic residues: Phe, Tyr, Trp.
  • the sequences of the catalytic sites ofL-asparaginases are highly conserved between Erwinia chrysanthemi, Erwinia carotovora, and E. coli L-asparaginase II (Id).
  • the active site flexible loop contains amino acid residues 14-33, and structural analysis show that Thrl5, Thr95, Ser62, Glu63, Asp96, and Alal20 contact the ligand (Id).
  • Aghaipour et al. have conducted a detailed analysis of the four active sites of Erwinia chrysanthemi L-asparaginase by examining high resolution crystal structures of the enzyme complexed with its substrates (Aghaipour (2001) Biochemistry 40, 5655-5664).
  • Kotzia et al. provide sequences for L-asparaginases from several species and subspecies of Erwinia and, even though the proteins have only about 75- 77% identity between Erwinia chrysanthemi and Erwinia carotovora, they each still have L- asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657-669).
  • Moola et al performed epitope mapping studies of Erwinia chrysanthemi 3937 L-asparaginase and were able to retain enzyme activity even after mutating various antigenic sequences in an attempt to reduce immunogenicity of the asparaginase (Moola (1994) Biochem. J. 302, 921-927).
  • fragments of the protein of SEQ ID NO: 1 are also comprised within the definition of the protein used in the L-asparaginase of the invention.
  • the term “a fragment of SEQ ID NO: l” means that the sequence of the polypeptide may include fewer amino-acids than the full-length SEQ ID NO: 1 but retains enough of the protein to confer Laminohydrolase activity.
  • a L-asparaginase has at least about 80% homology or identity with the protein comprising SEQ ID NO: 1.
  • the L-asparaginase of the formulation comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1. In some embodiments, each monomer unit has an amino acid sequences that is at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, or about 84% identical to SEQ ID NO: 1.
  • each monomer unit has an amino acid sequences that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90% about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1.
  • the L-asparaginase of the formulation is present at a concentration of about 20 mg/mL.
  • the L-asparaginase of the formulation is non- PEGylated. In some embodiments, the L-asparaginase of the formulation is non-PASylated.
  • the L-asparaginase of the formulation is non-PEGylated and non- PASylated.
  • the L-asparaginase formulation comprises one or more stabilizers.
  • the one or more stabilizers comprise one or more disaccharides, one or more sorbitols, one or more amino acids, or any combination thereof.
  • the L- asparaginase formulation comprises one or more buffers.
  • the one or more buffers comprise acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane (e.g., Tris), or any combination thererof.
  • the L-asparaginase formulation comprises one or more surfactants.
  • the one or more surfactants comprise polysorbate 80 (e.g.,Tween 80), polysorbate 20 (e.g.,Tween 20), poloxamer 188, or any combination thereof.
  • the L-asparaginase formulation comprises one or more polyol (e.g., mannitol and sorbitol), one or more disaccharides (e.g., sucrose and trehalose), and one or more polysaccharides (e.g., dextran 40).
  • the L-asparaginase formulation comprises sodium chloride.
  • the L-asparaginase formulation comprises one or more amino acids (e.g., proline, glycine, and arginie).
  • the formulation comprises one or more stabilizers at a concentration of between about 50 mM and about 300 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 75 mM and about 300 mM, between about 100 mM and about 300 mM, between about 125 mM and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM and about 300 mM.
  • the formulation comprises one or more stabilizers at a concentration of between about 50 mM and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM and about 225 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, or between about 50 mM and about 75 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 150 mM and about 275 mM.
  • the formulation comprises one or more stabilizers at a concentration of between about 150 mM and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM and about 180 mM, between 150 mM and about 170 mM, or between about 160 mM and 150 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 160 mM and about 200 mM, between about 170 mM and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 170 mM.
  • the one or more disaccharides of the formulation comprise trehalose. In other embodiments, the one or more disaccharides of the formulation comprise sucrose. In some embodiments, the one or more disaccharides of the formulation comprise a combination of trehalose and sucrose. [0050] In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 50 mM and about 300 mM.
  • the formulation comprises one or more disaccharides at a concentration of between about 75 mM and about 300 mM, between about 100 mM and about 300 mM, between about 125 mM and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM and about 300 mM.
  • the formulation comprises one or more disaccharides at a concentration of between about 50 mM and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM and about 225 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, or between about 50 mM and about 75 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 150 mM and about 275 mM.
  • the formulation comprises one or more disaccharides at a concentration of between about 150 mM and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM and about 180 mM, between 150 mM and about 170 mM, or between about 160 mM and 150 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 160 mM and about 200 mM, between about 170 mM and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 170 mM.
  • the formulation comprises trehalose at a concentration of between about 50 mM and about 300 mM.
  • the formulation comprises one or more disaccharides at a concentration of between about 75 mM and about 300 mM, between about 100 mM and about 300 mM, between about 125 mM and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM and about 300 mM.
  • the formulation comprises trehalose at a concentration of between about 50 mM and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM and about 225 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, or between about 50 mM and about 75 mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 150 mM and about 275 mM.
  • the formulation comprises trehalose at a concentration of between about 150 mM and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM and about 180 mM, between 150 mM and about 170 mM, or between about 160 mM and 150 mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 160 mM and about 200 mM, between about 170 mM and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 170 mM.
  • the one or more buffers of the formulation are substantially free of amino acid. In some embodiments, the one or more buffers of the formulation are substantially free of histidine or arginine, or both. In some embodiments, the one or more buffers of the formulation are substantially free of histidine. In some embodiments, the one or more buffers of the formulation comprise a phosphate buffer, an acetate buffer, or any combination thereof. In some embodiments, the one or more buffers comprise a phosphate buffer. In some embodiments, the one or more buffers comprise an acetate buffer. In some embodiments, the one or more buffers comprise a combination of a phosphate buffer and an acetate buffer.
  • the one or more buffers comprise an acetate salt of an alkali metal. In some embodiments, the one or more buffers comprise a phosphate salt of an alkali metal. In some embodiments, the one or more buffers comprise sodium phosphate. [0053] In some embodiments, formulation comprises one or more buffers at a concentration of between about 0.5 mM and between about 50 mM.
  • formulation comprises one or more buffers at a concentration of between about 1 mM and about 50 mM, between about 2.5 mM and about 50 mM, between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 mM, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM.
  • formulation comprises one or more buffers at a concentration of between about 0.5 mM and about 45 mM, between about 0.5 mM and about 40 mM, between about 0.5 mM and about 35 mM, between about 0.5 mM and about 30 mM, between about 0.5 mM and 25 mM, between about 0.5 mM and about 20 mM, between about 0.5 mM and about 15 mM, between about 0.5 mM and about 10 mM, between about 0.5 mM and about 5 mM, between about 0.5 M and about 2.5 mM, or between 0.5 mM and about 1 mM.
  • formulation comprises one or more buffers at a concentration of between about 10 mM and about 30 mM. In some embodiments, formulation comprises one or more buffers at a concentration of between about 15 mM and about 25 mM. In some embodiments, formulation comprises one or more buffers at a concentration of about 20 mM. [0054] In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 0.5 mM and between about 50 mM.
  • the formulation comprises sodium phosphate at a concentration of between about 1 mM and about 50 mM, between about 2.5 mM and about 50 mM, between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 mM, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM.
  • the formulation comprises sodium phosphate at a concentration of between about 0.5 mM and about 45 mM, between about 0.5 mM and about 40 mM, between about 0.5 mM and about 35 mM, between about 0.5 mM and about 30 mM, between about 0.5 mM and 25 mM, between about 0.5 mM and about 20 mM, between about 0.5 mM and about 15 mM, between about 0.5 mM and about 10 mM, between about 0.5 mM and about 5 mM, between about 0.5 M and about 2.5 mM, or between 0.5 mM and about 1 mM.
  • the formulation comprises sodium phosphate at a concentration of between about 10 mM and about 30 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 15 mM and about 25 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 20 mM. [0055] In some embodiments, the formulation further comprises an alkali metal salt. In some embodiments, the formulation further comprises a halide salt. In some embodiments, the formulation further comprises a halide salt of an alkali metal. In some embodiments, the formulation further comprises sodium chloride. In some embodiments, the formulation further comprises sodium chloride at a concentration of between about 25 mM and about 150 mM.
  • the formulation further comprises sodium chloride at a concentration of between about 30 mM and about 150 mM, between about 40 mM and about 150 mM, between about 50 mM and about 150 mM, between about 60 mM and about 150 mM, between about 70 mM and about 150 mM, between about 80 mM and about 150 mM, between about 90 mM and about 150 mM, between about 100 mM and about 150 mM, between about 110 mM and about 150 mM, between about 120 mM and about 150 mM, between about 130 mM and about 150 mM, or between about 140 mM and about 150 mM.
  • the formulation comprises sodium chloride at a concentration of between about 25 mM and about 140 mM, between about 25 mM and 130 mM, between about 25 mM and about 120 mM, between about 25 mM and about 110 mM, between about 25 mM and about 100 mM, between about 25 mM and about 90 mM, between about 25 mM and about 80 mM, between about 25 mM and about 70 mM, between about 25 mM and about 60 mM, between about 25 mM and about 50 mM, between about 25 mM and about 40 mM, or between about 25 mM and 30 mM.
  • the formulation comprises sodium chloride at a concentration of between about 30 mM and about 70 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of between about 40 mM and about 60 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of about 50 mM. [0056] In some embodiments, the formulation further comprises one or more excipients. In some embodiments, the one or more excipients comprise one or more surfactants. In some embodiments, the one or more excipients comprise one or more emulsifiers. In some embodiments, the one or more excipients are derived from sorbitan.
  • the one or more excipients comprise polysorbate 80 (also known as PS- 80). In some embodiments, the one or more excipients comprise polysorbate 20. In some embodiments, the one or more excipients comprise a combination of polysorbate 80 and polysorbate 20. [0057] In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.3% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.28% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.26% (w/v).
  • the formulation comprises one or more excipients at a concentration of no more than about 0.24% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.22% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.3% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.3% (w/v). [0058] In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.2% (w/v).
  • the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.18% (w/v), between about 0.004% (w/v) and about 0.16% (w/v), between about 0.004% (w/v) and about 0.14% (w/v), between about 0.004% (w/v) and about 0.12% (w/v), between about 0.004% (w/v) and about 0.1% (w/v), between about 0.004% (w/v) and about 0.08% (w/v), between about 0.004% (w/v) and about 0.06% (w/v), between about 0.004% (w/v) and about 0.04% (w/v), between about 0.004% (w/v) and about 0.03%, between about 0.004% (w/v) and about 0.025% (w/v), between about 0.004% (w/v) and about 0.02% (w/v), between about 0.004% (w/v) and about 0.015% (w/v),
  • the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.2% (w/v), between about 0.015% (w/v) and about 0.2% (w/v), between about 0.02% (w/v) and about 0.2% (w/v), between about 0.025% (w/v) and about 0.2% (w/v), between about 0.03% (w/v) and about 0.2% (w/v), between about 0.04% (w/v) and about 0.2% (w/v), between about 0.05% (w/v) and about 0.2% (w/v), between about 0.06% (w/v) and about 0.2% (w/v), between about 0.07% (w/v) and about 0.2% (w/v), between about 0.08% (w/v) and about 0.2% (w/v), between about 0.09% (w/v) and about 0.2% (w/v), between about 0.1% (w/v) and about 0.2% (w/v), between about 0.12% (w/v/v),
  • the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.03% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.015% (w/v) and about 0.025% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.02% (w/v). [0059] In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.004% (w/v) and about 0.2% (w/v).
  • the formulation further comprises polysorbate 80 at a concentration of between about 0.004% (w/v) and about 0.18% (w/v), between about 0.004% (w/v) and about 0.16% (w/v), between about 0.004% (w/v) and about 0.14% (w/v), between about 0.004% (w/v) and about 0.12% (w/v), between about 0.004% (w/v) and about 0.1% (w/v), between about 0.004% (w/v) and about 0.08% (w/v), between about 0.004% (w/v) and about 0.06% (w/v), between about 0.004% (w/v) and about 0.04% (w/v), between about 0.004% (w/v) and about 0.03%, between about 0.004% (w/v) and about 0.025% (w/v), between about 0.004% (w/v) and about 0.02% (w/v), between about 0.004% (w/v) and about 0.015% (w/v), or between about
  • the formulation further comprises polysorbate 80 at a concentration of between about 0.01% (w/v) and about 0.2% (w/v), between about 0.015% (w/v) and about 0.2% (w/v), between about 0.02% (w/v) and about 0.2% (w/v), between about 0.025% (w/v) and about 0.2% (w/v), between about 0.03% (w/v) and about 0.2% (w/v), between about 0.04% (w/v) and about 0.2% (w/v), between about 0.05% (w/v) and about 0.2% (w/v), between about 0.06% (w/v) and about 0.2% (w/v), between about 0.07% (w/v) and about 0.2% (w/v), between about 0.08% (w/v) and about 0.2% (w/v), between about 0.09% (w/v) and about 0.2% (w/v), between about 0.1% (w/v) and about 0.2% (w/v), between about 0.12% (w/v) and
  • the formulation further comprises polysorbate 80 at a concentration of between about 0.01% (w/v) and about 0.03% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.015% (w/v) and about 0.025% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.02% (w/v). [0060] In some embodiments, the formulation provided herein has a pH of between about 4.0 and about 8.5.
  • the formulation has a pH of between about 4.5 and about 8.5, between about 5.0 and about 8.5, between about 5.5 and about 8.5, between about 6.0 and about 8.5, between about 6.5 and about 8.5, between about 7.0 and about 8.5, between about 7.5 and about 8.5, or between about 8.0 and about 8.5. In some embodiments, the formulation has a pH of between about 4.5 and about 8.0, between about 4.5 and about 7.5, between about 4.5 and about 7.0, between about 4.5 and about 6.5, between about 4.5 and about 6.0, between about 4.5 and about 5.5, or between about 4.5 and about 5.0.
  • the formulation provided herein has a pH of between about 4.0 and about 8.0, between about 4.0 and about 7.5, between about 4.0 and about 7.0, between about 4.0 and about 6.5, between about 4.0 and about 6.0, between about 4.0 and about 5.5, between about 4.0 and about 5.0, or between about 4.0 and about 4.5. In some embodiments, the formulation provided herein has a pH of between about 5.5 and about 8.5. In some embodiments, the formulation provided herein has a pH of between about 6.0 and about 8.0. In some embodiments, the formulation provided herein has a pH of between 6.5 and about 7.5. In some embodiments, the formulation provided herein has a pH of around 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L- asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
  • the L-asparaginase formulation comprises one or more stabilizers.
  • the one or more stabilizers comprise one or more disaccharides, one or more sorbitols, one or more amino acids, or any combination thereof.
  • the L-asparaginase formulation comprises one or more disaccharides. In some embodiments, the one or more disaccharides comprise trehalose, sucrose, or any combination thereof. In some embodiments, the L-sparaginase formulation comprises one or more buffers. In some embodiments, the one or more buffers, wherein the one or more buffers are substantially free of amino acid. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 °C for two months. In some embodiments, the L-asparaginase formulation comprises less than 2% high-molecular- weight (HMW) species after storage at 40 °C for two months.
  • LMW low-molecular-weight
  • HMW high-molecular- weight
  • the L- asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species and less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and is present at a concentration of around 20 mg/mL; (ii) one or more stabilizers (e.g., disaccharides, sorbitols, amino acids, or any combination thereof) at a concentration between about 50 mM and about 300 mM; (iii) one or more buffers (e.g., acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane, or combination thereof) at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM; and (v) one or more surfactants (e.g., polysorbate 80, poly
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, in conjunction with embodiments above or below, the formulation has a pH of about 7.0.
  • the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration between about 50 mM and about 300 mM; (iii) sodium phosphate, wherein the sodium phosphate (e.g., dibasic anhydrous, monobasic monohydrate, or combination thereof) is present at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM and
  • the L-asparaginase in conjunctions with embodiments above or below, can be formulated in a unit dosage form.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • a unit dosage can be administered every 12 hours, every 24 hours, every 48 hours, or every 72 hours. In some embodiments, a unit dosage is administered every 48 hours.
  • a unit dosage contain from about 5 mg to about 50 mg (e.g., about 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, and 49 mg) of the L- asparaginase.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid; and (iv) sodium chloride, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid; and (iv) one or more excipients, wherein the formulation comprises less than about 5% low-molecular- weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular- weight
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid; (iv) sodium chloride; and (v) one or more excipients, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid; and (iv) polysorbate 80, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid; (iv) sodium chloride; and (v) polysorbate 80, wherein the formulation comprises less than about 5% low- molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low- molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) trehalose; (iii) sodium phosphate; (iv) sodium chloride; and (v) polysorbate 80, wherein the formulation comprises less than about 5% low- molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low- molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration between about 50 mM and about 300 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM;
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • a formulation as described elsewhere herein wherein the formulation comprises less than about 5% low-molecular- weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 4.5% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 4% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • the formulation comprises less than about 3.5% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 3% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 2.5% low- molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 2% low-molecular-weight (LMW) species after storage at 37 °C for one week. the formulation comprises less than about 1.5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • the formulation comprises less than about 1% low-molecular-weight (LMW) species after storage at 37 °C for one week. In some embodiments, the formulation comprises less than about 0.5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the formulation comprises less than about 0.5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • the L-asparaginase formulations of the present disclosure can be used in the treatment of a disease in a subject (for example, a human), wherein the disease is treatable by the depletion of asparagine or the administration of asparaginase.
  • a subject for example, a human
  • the disease is treatable by the depletion of asparagine or the administration of asparaginase.
  • the human subject has, prior to administration of the L-asparaginase, experienced silent inactivation of the E. Coli-derived asparaginase.
  • the subject has, prior to administration of the L-asparaginase, experienced an allergic reaction to the E. Coli- derived asparaginase.
  • the subject has, prior to administration of the L- asparaginase, experienced anaphylaxis to the E. Coli-derived asparaginase.
  • objective signs of allergy or hypersensitivity include testing “antibody positive” for an asparaginase enzyme.
  • the L-asparaginase formulations of the present disclosure are useful in the treatment or the manufacture of a medicament for use in the treatment of acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • the incidence of relapse in ALL patients following treatment with L-asparaginase remains high, with approximately 10-25% of pediatric ALL patients having early relapse (e.g., some during maintenance phase at 30-36 months post-induction). If a patient treated with E.
  • the L-asparaginase of the invention may be used in a method of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-derived asparaginases.
  • L-asparaginase formulations of the present disclosure are useful in treating include, but are not limited to, the following: malignancies, or cancers, including but not limited to, hematalogic malignancies, lymphoma, non- Hodgkin’s lymphoma, NK lymphoma, pancreatic cancer, Hodgkin’s disease, large cell immunoblastic lymphoma, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), biphenotypic B-cell myelomonocytic Leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma, and diffuse large B-cell lymphoma (DLBCL).
  • malignancies or cancers, including but not limited to, hematalogic
  • L-asparaginase formulations useful in treating are cancers including, but not limited to, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforma and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate carcinoma,
  • cancers including
  • the cancer may be a solid cancer, for example lung cancer or breast cancer.
  • Representative non-malignant hematologic diseases which respond to asparagine depletion include immune system-mediated blood diseases, including, but not limited to, infectious diseases such as those caused by HIV infection (i.e., AIDS).
  • Nonhematologic diseases associated with asparagine dependence include autoimmune diseases, for example, rheumatoid arthritis, collagen vascular diseases, AIDS, osteoarthritis, Issac’s syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus (SLE), rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn’s disease), primary billiary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves’ disease.
  • autoimmune diseases for example, rheumatoid arthritis, collagen vascular diseases, AIDS, osteoarthritis, Issac’s syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic l
  • the disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof comprises WNT mutated colorectal cancer (CRC).
  • the disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof comprises relapse remitting (R/R) acute myeloid leukemia (AML).
  • L-asparaginase formulations of the present disclosure are useful in treating include, but are not limited to, sarcoma, breast cancer, metastatic breast cancer, liver cancer, stomach cancer, colorectal cancer, and head and neck cancer.
  • Pharmaceutical compositions and co-administration [0080] In some embodiments, treatment with a L-asparaginase formulation of the present disclosure is co-administered with a multi-agent chemotherapeutic regimen. In some embodiments, treatment with a L-asparaginase formulation of the present disclosure is co- administered with one or more other chemotherapeutic agents as part of a multi-agent chemotherapeutic regimen.
  • treating patients with a L-asparaginase formulation of the present disclosure in addition to other agents helps to ensure availability of an asparaginase for patients who have developed hypersensitivity to E. coli derivedasparaginase.
  • agents that may be part of a multi-agent chemotherapeutic regimen with a L-asparaginase of the present disclosure include, but are not limited to: cytarabine, vincristine, daunorubicin, methotrexate, leuvocorin, doxorubicin, anthracycline, corticosteroids and glucocortiods (including but not limited to prednisone, prednisolone, and/or dexamethasone), cyclophosphamide, 6-mercaptopurine, venetoclax, and etoposide.
  • the multi-agent chemotherapeutic regimen is the L-asparaginase and one additional chemotherapeutic agent. In some embodiments, the multi-agent chemotherapeutic regimen is the L-asparaginase and two or more additional chemotherapeutic agents. [0082] As an example, patients with ALL will be co-administered the L- asparaginase of the present disclosure along with a multi-agent chemotherapy during 3 chemotherapy phases including induction, consolidation or intensification, and maintenance.
  • the L-asparaginase formulation of the present disclosure is co- administered with an asparagine synthetase inhibitor (e.g., such as set forth in WO 2007/103290, which is herein incorporated by reference in its entirety).
  • an asparagine synthetase inhibitor e.g., such as set forth in WO 2007/103290, which is herein incorporated by reference in its entirety.
  • the L-asparaginase formulation of the present disclosure is not co-administered with an asparagine synthetase inhibitor, but is co-administered with other chemotherapy drugs.
  • the L-asparaginase formulation of the present disclosure is co- administered with an asparagine synthetase inhibitor and other chemotherapy drugs.
  • the L- asparaginase formulation of the present disclosure can be co-administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen.
  • the L-asparaginase of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens and, more specifically, the L- asparaginase comprising the sequence of SEQ ID NO: 1.
  • the formulations described herein can be administered to a patient as a pharmaceutical composition using standard techniques. Techniques and formulations generally may be found in Remington’s Pharmaceutical Sciences, 22nd edition, Mack Publishing, 2015 (herein incorporated by reference).
  • Suitable dosage forms depend upon the use or the route of entry, for example, oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the therapeutic agent to reach a target cell or otherwise have the desired therapeutic effect.
  • pharmaceutical compositions injected into the blood stream preferably are soluble.
  • the pharmaceutical compositions according to the present disclosure can be formulated as pharmaceutically acceptable salts and complexes thereof.
  • Pharmaceutically acceptable salts are non-toxic salts present in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate pharmaceutical use by altering the physical characteristics of the compound without preventing it from exerting its physiological effect.
  • Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administering higher concentrations of the drug.
  • the pharmaceutically acceptable salt of a modified protein as described herein may be present as a complex, as those in the art will appreciate.
  • Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate, and quinate.
  • Pharmaceutically acceptable salts can be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
  • acids including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid.
  • Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present.
  • acidic functional groups such as carboxylic acid or phenol are present.
  • Such salts can be prepared using the appropriate corresponding bases.
  • Pharmaceutically acceptable carriers and/or excipients can also be incorporated into a pharmaceutical composition according to the invention to facilitate administration of the particular asparaginase.
  • compositions according to the invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration.
  • the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops.
  • injection parenteral administration
  • pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank’s solution, or Ringer’s solution.
  • the L-asparaginase formulation is administered intramuscularly.
  • the L-asparaginase formulation is administered intravenously.
  • the L-asparaginase formulation is administered subcutaneously.
  • Systemic administration can also be accomplished by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration for example, may be through nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories.
  • a dose is an amount administered to the human subject over a certain time and frequency.
  • the dose of L-asparaginase formulation will be given to a human subject with hypersensitivity only when the hypersensitivity subsides.
  • a L-asparaginase formulation is administered to a human subject in an amount from about 10 mg/m 2 to 100 mg/m 2 .
  • a L-asparaginase formulation is administered intramuscularly every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 25 mg/m 2 .
  • a L-asparaginase formulation is administered intravenously every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 37.5 mg/m 2 .
  • a L-asparaginase formulation is administered intravenously every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 50 mg/m 2 .
  • the L-asparaginase formulation administered in such doses is not conjugated to a polymer such as a PEG moiety and/or is not conjugated to a peptide comprising solely alanine and/or proline residues.
  • Dose amount [0090] The amounts of the L-asparaginase formulation of the present disclosure that are to be delivered will depend on many factors, for example, the IC50, EC50, the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated. The importance of these and other factors to be considered are well known to those of ordinary skill in the art.
  • the amount of the L-asparaginase formulation of the present disclosure will be administered at a range from about 1 milligram per square meter of the surface area of the patient's body (mg/m 2 ) to 1,000/m 2 , with a dosage range of about 10 mg/m 2 to about 100 mg/m 2 to treat disease, including but not limited to ALL or LBL. Of course, other dosages and/or treatment regimens may be employed, as determined by the attending physician. [0091] In some embodiments, the method comprises administering the recombinant L- asparaginase of the present disclosure at an amount from about 10 mg/m 2 and about 100 mg/m 2 .
  • the method comprises administering the L- asparaginase formulation of the present disclosure at an amount from 10 mg/m 2 and 100 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount of about 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 95, or 95 mg/m 2 or an equivalent amount thereof (for example on a protein content basis).
  • the L-asparaginase formulation of the present disclosure is administered at an amount selected from the group consisting of about 10, 20, 30, 40, 50, 60, 70, 80, 90, and about 100 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered at a dose more than or equal to about 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 95, 100, 200, or 300 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered at a dose less than or equal to about 300, 200100, 95, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 mg/m 2 .
  • the L- asparaginase formulation of the present disclosure is administered in an amount between about 12 mg/m 2 and about 90 mg/m 2 .
  • the recombinant L- asparaginase of the present disclosure is administered in an amount between about 20 mg/m 2 and about 80 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m 2 and about 70 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m 2 and about 80 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 37.5 mg/m 2 and about 80 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 37.5 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 25 mg/m 2 and about 37.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m 2 and about 100 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m 2 and about 65 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m 2 and about 80 mg/m 2 .
  • the method comprises administering the recombinant L- asparaginase of the present disclosure at an amount from about 25 mg/m 2 and about 50 mg/m 2 .
  • the method comprises administering the L- asparaginase formulation of the present disclosure at an amount from 25 mg/m 2 and 50 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount of about 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, or 50 mg/m 2 or an equivalent amount thereof (for example on a protein content basis).
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 20 mg/m 2 and about 30 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 22.5 mg/m 2 and about 28.5 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 23 mg/m 2 and about 27 mg/m 2 . In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 24 mg/m 2 and about 26 mg/m 2 . In an exemplary embodiment, the L- asparaginase formulation of the present disclosure is administered in an amount between about 24.5 mg/m 2 and about 25.5 mg/m 2 . [0094] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 60 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 42.5 mg/m 2 and about 58.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 43 mg/m 2 and about 57 mg/m 2 . In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 44 mg/m 2 and about 56 mg/m 2 . In an exemplary embodiment, the L- asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m 2 and about 55 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 46 mg/m 2 and about 54 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 47.5 mg/m 2 and about 52.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 48 mg/m 2 and about 52 mg/m 2 . In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 49 mg/m 2 and about 51 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 49.5 mg/m 2 and about 50.5 mg/m 2 . [0095] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m 2 and about 70 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 65 mg/m 2 .
  • the recombinant L- asparaginase of the present disclosure is administered in an amount between about 45 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L- asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m 2 and about 55 mg/m 2 . [0096] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m 2 and about 70 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 55 mg/m 2 and about 60 mg/m 2 . [0097] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m 2 and about 55 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 47.5 mg/m 2 and about 50 mg/m 2 . [0098] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m 2 and about 35 mg/m 2 . [0099] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m 2 and about 95 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m 2 and about 90 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 85 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m 2 and about 80 mg/m 2 . In an exemplary embodiment, the L- asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 55 mg/m 2 and about 70 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 60 mg/m 2 and about 65 mg/m 2 . [0100] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m 2 and about 55 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 50 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 42.5 mg/m 2 and about 57.5 mg/m 2 . [0101] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m 2 and about 70 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m 2 and about 65 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L- asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m 2 and about 55 mg/m 2 . [0102] In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 10 mg/m 2 and about 50 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 12.5 mg/m 2 and about 47.5 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 15 mg/m 2 and about 45 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 20 mg/m 2 and about 42.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 22.5 mg/m 2 and about 40 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 24 mg/m 2 and about 39 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 27 mg/m 2 and about 37.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 30 mg/m 2 and about 45 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 25 mg/m 2 .
  • the recombinant L- asparaginase of the present disclosure is administered intramuscularly in an amount of 25 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 25 mg/m 2 and about 80 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 37.5 mg/m 2 and about 80 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 37.5 mg/m 2 and about 65 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 25 mg/m 2 and about 37.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 30 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 35 mg/m 2 and about 70 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m 2 and about 65 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 50 mg/m 2 and about 55 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 mg/m 2 and about 70 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 50 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 55 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 mg/m 2 and about 55 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 47.5 mg/m 2 and about 50 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 30 mg/m 2 and about 35 mg/m 2 . [0103] In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 10 mg/m 2 and about 95 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 20 mg/m 2 and about 60 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 22.5 mg/m 2 and about 57.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 25 mg/m 2 and about 55 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 27.5 mg/m 2 and about 47.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 30 mg/m 2 and about 45 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 32.5 mg/m 2 and about 42.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 21.5 mg/m 2 and about 38.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 36 mg/m 2 and about 45 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 37.5 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 37.5 mg/m 2 . In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 50 mg/m 2 . [0104] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m 2 and about 37.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m 2 and about 100 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25mg/m 2 and about 80 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m 2 and about 35 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m 2 and about 95 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m 2 and about 90 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m 2 and about 85 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 45 mg/m 2 and about 80 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 50 mg/m 2 and about 75 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 55 mg/m 2 and about 70 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 60 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m 2 and about 55 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m 2 and about 50 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 42.5 mg/m 2 and about 57.5 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m 2 and about 75 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m 2 and about 70 mg/m 2 .
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m 2 and about 65 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 45 mg/m 2 and about 60 mg/m 2 . In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 50 mg/m 2 and about 55 mg/m 2 .
  • the method comprises administering a recombinant L- asparaginase of the present disclosure that elicits a lower immunogenic response in a patient compared to a non-recombinant L-asparaginase.
  • treatment will be administered at a dose ranging from about 1 mg/m 2 to about 1000 mg/m 2 , typically about 10 mg/m 2 to about 100 mg/m 2 , at a schedule ranging from about three a week to about once a month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracycline.
  • chemotherapy drugs including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide,
  • the L-asparaginase formulation of the present disclosure can be administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen.
  • the L-asparaginase formulation of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens, and more specifically, the L-asparaginase formulation comprises a sequence according to SEQ ID NO:1.
  • the L-asparaginase formulation of the present disclosure is administered at a dose that depletes L-asparagine to undetectable levels using methods and apparatus known in the art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) for a single dose.
  • the L-asparaginase formulation of the present disclosure is administered three times a week.
  • the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days.
  • the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week.
  • the L-asparaginase formulation of the present disclosure is administered three times a week for at least one to three weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for about one to three weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the week for about one to three weeks. [0109] In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week for about two weeks.
  • the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for about two weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week for about two weeks. [0110] In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week for two weeks. In some embodiments, the L- asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for two weeks.
  • the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week for two weeks. [0111] In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week, continuing until the patient no longer has a disease that is treatable by depletion of asparagine. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, continuing until the patient no longer has a disease that is treatable by depletion of asparagine.
  • the recombinant L- asparaginase of the present disclosure is administered on Monday, Wednesday, and Friday of the same week, continuing until the patient no longer has a disease that is treatable by depletion of asparagine.
  • the L-asparaginase formulation of the present disclosure is administered three times a week, continuing until the patient decides to end or postpone treatment.
  • the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, continuing until the patient decides to end or postpone treatment.
  • the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week, continuing until the patient decides to end or postpone treatment. [0113] In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every 40 to 58 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 42 to 56 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 44 to 52 hours. In some embodiments, the L- asparaginase formulation of the present disclosure is administered about every 46 to 50 hours.
  • the recombinant L- asparaginase of the present disclosure is administered about every 72 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every 64 to 80 hours. In some embodiments, the L- asparaginase formulation of the present disclosure is administered about every 66 to 78 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 68 to 76 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 70 to 74 hours. [0114] In some embodiments, L-asparaginase formulation of the present disclosure is administered as a second line therapy with patients who are hypersensitive to an E.
  • the L-asparaginase formulation is administered to the human subject as a substitute for a dose of a long-acting E. coli-derived asparaginase.
  • six doses of the L-asparaginase formulation are administered to the human subject as a substitute for one dose of the long-acting E. coli-derived asparaginase.
  • seven doses of the L-asparaginase formulation are administered to the human subject as a substitute for one dose of the long-acting E.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle comprises a first dose, a second dose, and a third dose, wherein the cycle is optionally repeatable, and wherein the first dose, second dose, and third dose are administered about 48-72 hours apart.
  • dose amounts may vary within the cycle.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 25 mg/m 2 , the second dose of the cycle is 25 mg/m 2 and the third dose of the cycle is 50 mg/m 2 , followed by the rest period of two consecutive days.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 25 mg/m 2 , the second dose of the cycle is 25 mg/m 2 and the third dose of the cycle is 37.5 mg/m 2 , followed by the rest period of two consecutive days.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m 2 , the second dose of the cycle is 37.5 mg/m 2 and the third dose of the cycle is 37.5 mg/m 2 , followed by the rest period of two consecutive days.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m 2 , the second dose of the cycle is 25 mg/m 2 and the third dose of the cycle is 37.5 in mg/m 2 , followed by the rest period of two consecutive days.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m 2 , the second dose of the cycle is 25 mg/m 2 and the third dose of the cycle is 25 mg/m 2 , followed by the rest period of two consecutive days.
  • the first dose of the cycle is administered on a Monday
  • the second dose of the cycle is given on a Wednesday
  • the third dose of the cycle is given on a Friday.
  • a dose regimen for the L-asparaginase formulation comprises a cycle, where the cycle is optionally repeatable, and where the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, where the first dose of the cycle is 25 mg/m 2 , the second dose of the cycle is 25 mg/m 2 and the third dose of the cycle is 50 mg/m 2 , followed by the rest period of two consecutive days.
  • the first dose of the cycle is administered on a Monday
  • the second dose of the cycle is given on a Wednesday
  • the third dose of the cycle is given on a Friday.
  • the present disclosure provides a method for depleting asparaginase in a human subject to treat Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LBL), the method comprising: on Mondays, Wednesdays, and Fridays, administering intramuscularly about 25 mg/m 2 of L-asparaginase to the human subject, such that the human subject receives a total of seven doses of L-asparaginase over a two week period.
  • ALL Acute Lymphoblastic Leukemia
  • LBL Lymphoblastic Lymphoma
  • the present disclosure provides a method for depleting asparaginase in a human subject to treat Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LBL), the method comprising: (a) on Mondays and Wednesdays, administering intramuscularly about 25 mg/m 2 of L-asparaginase to the human subject, and (b) on Fridays, administering intramuscularly about 50 mg/m 2 of L-asparaginase to the human subject; such that the human subject receives a total of six doses of L-asparaginase over a two week period.
  • ALL Acute Lymphoblastic Leukemia
  • LBL Lymphoblastic Lymphoma
  • the dose regimen may encompass any number of cycles for any number of weeks or until any endpoint that is specified herein.
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of (i) 25 mg/m 2 on Monday, (ii) 25 mg/m 2 on Wednesday, and (iii) 50 mg/m 2 on Friday.
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 25 mg/m 2 every 48 hours.
  • the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 37.5 mg/m 2 every 48 hours.
  • the L- asparaginase formulation of the present disclosure is administered intravenously in an amount of 50 mg/m 2 every 48 hours. [0129] In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 25 mg/m 2 every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 37.5 mg/m 2 every 48 hours. In some embodiments, the L- asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 50 mg/m 2 every 48 hours.
  • the L-asparaginase formulation of the present disclosure is administered (i) intravenously in an amount of 25 mg/m 2 on Monday, (ii) intravenously in an amount of 25 mg/m 2 on Wednesday, and (iii) intramuscularly in an amount of 50 mg/m 2 on Friday.
  • a dosing regimen may comprise administration of six doses (e.g., a first dose, a second dose, a third dose, a fourth dose, a fifth dose, and a sixth dose) of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously or intramuscularly in an amount of 25 mg/m 2 or 37.5 mg/m 2 on Monday, (ii) a second dose is administered intravenously or intramuscularly in an amount of 25 mg/m 2 37.5 mg/m 2 on Wednesday, and (iii) a third dose is administered intravenously or intramuscularly in an amount of 25 mg/m 2 37.5 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is
  • a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (ii) a second dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 25 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m 2 on Friday.
  • a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intramuscularly in an amount of 25 mg/m 2 on Monday, (ii) a second dose is administered intramuscularly in an amount of 25 mg/m 2 on Wednesday, and (iii) a third dose is administered intramuscularly in an amount of 25 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m 2 on Friday.
  • a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 37.5 mg/m 2 on Monday, (ii) a second dose is administered intravenously in an amount of 37.5 mg/m 2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 37.5 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m 2 on Friday.
  • a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intramuscularly in an amount of 37.5 mg/m 2 on Monday, (ii) a second dose is administered intramuscularly in an amount of 37.5 mg/m 2 on Wednesday, and (iii) a third dose is administered intramuscularly in an amount of 37.5 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m 2 on Friday.
  • a dosing regimen comprise administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (ii) a second dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 50 mg/m 2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m 2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m 2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m 2 on Friday.
  • kits comprising: (i) any one of the formulations described herein; and (ii) instructions for treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof.
  • the kit comprises a formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
  • the L-asparaginase formulation comprises less than about 0.6% low- molecular-weight (LMW) species after storage at 40 °C for two months. In some embodiments, the L-asparaginase formulation comprises less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months. In some embodiments, the L- asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species and less than 2% high-molecular-weight (HMW) species after storage at 40 °C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v), wherein the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v
  • the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7.0.
  • the kits comprise instructions for treating cancer. In some embodiments, the kits comprise instructions for treating acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is relapsed. In some embodiments, the kits comprise instructions for treating lymphoblastic lymphoma (LBL). In some embodiments, the LBL is relapsed.
  • ALL acute lymphoblastic leukemia
  • LBL lymphoblastic lymphoma
  • Nessler Activity was measured by Nessler assay, in which the measurement of asparaginase activity is based on an endpoint limit assay in which the enzyme is incubated at +37 °C under saturating L-asparagine concentration for 15 minutes. The reaction is stopped by addition of Nessler’s reagent and the amount of ammonia produced during the reaction is assessed colorimetrically (at 450 nm) using a calibration curve constructed from known quantities of ammonium sulfate.
  • SEC Size Exclusion Chromatography
  • Example 1 Baseline Buffer/Excipient Screen
  • the effects of pH and buffer type from six buffer/pH combinations in the range of 4.5 – 8.0 were assessed on stability of L-asparaginase.
  • An additional set of formulations with 6 different excipients in either 20 mM histidine buffer at pH 7.0 or 20 mM sodium phosphate at pH 8.0 were prepared as an initial excipient screen (Table 1).
  • L- asparaginase 1.0 mL of Lot RM-LAP-P03/P05/P06 Pool of Bulks at 20.9 mg/mL
  • excipients proline, lysine, sodium chloride, arginine, trehalose, sorbitol, and sucrose in histidine buffer at pH 7.0 or phosphate buffer at pH 8.0 were evaluated for the influence of excipients.
  • SEC and the Nessler activity assay were used to assess the stability of RC in the presence of these excipients in the selected buffers.
  • the purity of 26 formulations was assessed by SEC-HPLC. Results are summarized in Table 2 and Table 3. Little difference was observed in the SEC profiles between the 5°C and 37°C conditions after 1 week storage for all formulations. However, differences were observed between the different formulations. The main peak purities had a range of 66.4 – 98.3%.
  • the histidine buffer formulation all had purity levels between 66.4% and 74.0%, while the remaining formulations had purity levels between 90.6% and 98.3%.
  • succinate, citrate, phosphate with lysine, and phosphate with arginine buffers a LMW species was observed at ⁇ 11.6 minutes ranging from 1.3 – 32.4%.
  • the formulations in acetate, phosphate, and tris buffers showed between 0.2 – 0.6% LMW species.
  • Formulations in phosphate buffer with sodium chloride, trehalose, sorbitol, and sucrose showed similar levels of LMW species to the formulations without excipients.
  • Table 3 [0149] Based on the highest purity and lowest LMW species percentage, acetate and phosphate buffer were selected for further evaluation. Additionally, trehalose, sorbitol, and sodium chloride were chosen as the excipients to be evaluated in the DOE to assess their impact on formulation stability under more stringent stress conditions.
  • Example 2 DOE [0150] A statistical design of experiment (DOE) approach considering one numeric factor (pH) and one categorical factor (excipient) was used to evaluate the effects of pH and excipient for acetate and phosphate buffer in the range of 4.5 – 5.5 and 7.0 – 8.0, respectively, on L-asparaginase.
  • DOE A statistical design of experiment (DOE) approach considering one numeric factor (pH) and one categorical factor (excipient) was used to evaluate the effects of pH and excipient for acetate and phosphate buffer in the range of 4.5 – 5.5 and 7.0 – 8.0, respectively, on L-asparaginase.
  • the Nessler activity assay for formulations exposed to a single freeze/thaw cycle or stored for 2 weeks at 40°C had activities that ranged from 600 to 1270 ⁇ mol/min.mgE (Table 4 and Table 5). No significant loss of activity of the formulations was observed the thermal or freeze/thaw stress, and it is undetermined if the formulations with increased activity is a result of the formulation buffer or the variability of the Nessler Activity assay.
  • the formulations were analyzed by RP-HPLC. By RP-HPLC, all formulations in both acetate and phosphate buffer were determined to be 100% main peak after a single freeze/thaw stress, and 98% main peak and 2% minor peak after 2 weeks at 40°C.
  • Table 5 [0153] All formulations and stress conditions were analyzed by SEC-HPLC. Results are shown in Table 6. After thermal stress, the purity in phosphate buffered formulations ranged from 88.70% to 99.31%, the HMW peak ranged from 0.66% to 1.00%, the LMW shoulder ranged from none detected to 10.98%, and the LMW peak was none detected. At pH 7.0, all three excipients show similar SEC profiles after thermal stress, and the main peak percentage was 99% for each excipient.
  • Table 6 [0154] The decrease in main peak purity and increase in LMW shoulder percentage were fit to a linear model dependent on pH only with the maximum main peak purity observed at pH 7.0 (Table 7).
  • the off-design formulations at pH 8.5 also show formation of significant quantities of LMW shoulder after thermal stress (Table 8).
  • the formation of HMW species was found to be dependent on excipient, but not pH, with sorbitol having the overall largest percentage of HMW species.
  • Table 7 Table 8 [0155] Under freeze/thaw stress for the sodium phosphate formulations, the SEC- HPLC main peak purity range from 98.05% to 99.34%, the HMW peak ranged from 0.66% to 0.71%, and the LMW peak ranged from none detected to 1.28%.
  • Table 11 [0158] At pH 7.0, the main peak percentage was 62.1-63.1%, 59.3-59.9%, and 57.2-57.5% for sodium chloride, trehalose, and sorbitol, respectively. No trend was observed for the amount of basic species under thermal stress. A similar trend for pH was observed for the main peak and acidic species percentages for these formulations under freeze/thaw stress (Table 12), but little difference was observed between the excipients. Table 12 [0159] After thermal stress, the acetate buffer formulations purity ranged from 51.3% to 56.4%, the acidic species from 5.9% to 7.3%, and basic species from 37.2% to 41.8% (Table 11 above).
  • the main peak decreased with decreasing pH, and the main peak percentage was also dependent on the excipient type. At pH 5.0, the main peak percentage was 54.0%, 55.2%, and 52.1% for sorbitol, trehalose, and sodium chloride respectively. No trend was observed for the amount of basic species observed under thermal stress. Under freeze/thaw stress in sodium acetate buffers, a similar trend was observed with pH and excipient relative to the thermal stress, although the difference between formulations are relatively small (Table 12 above).
  • the IEX-HPLC results for the off-design formulations are found in Table 13 and Table 14. Table 13 Table 14 [0160] All formulations were colorless and clear after vialing.
  • the numerical optimization was performed on the analytical results from the 20 mM sodium phosphate DOE after 2 weeks of thermal stress.
  • the formulation optimization was performed to find the optimal formulation without any constraints on the pH or excipient.
  • the optimization parameters and results are found in Table 18, Table 19, Table 20, and Table 21.
  • the optimal formulation was calculated to be 20 mM sodium phosphate, 250 mM trehalose at pH 7.2. If the appearance of particulate in the formulation is not factor as a constraint in the optimization and all results were given the same importance, the optimal formulation was calculated to be 20 mM sodium phosphate, 150 mM sodium chloride at pH 7.0.
  • One vial from each formulation was held at 5°C for the initial time point testing (8 total) during the freeze-thaw and agitation evaluation.
  • One and three freeze-thaw cycles were performed independently on separate vials of each formulation. Each freeze-thaw cycle was frozen at -75 ⁇ 10°C for a minimum of 2 hours and thawed at room temperature for a minimum of 2 hours and until completely thawed.
  • Three vials from each formulation were required for the agitation evaluation (24 total).
  • One vial was agitated on a rotator at 60 rpm at ambient temperature for 24 hours, one vial for 48 hours under the same conditions, and one vial was placed next to the rotator for the duration of the study as a control.
  • Controls for each formulation were held at 5°C for the freeze/thaw stress and room temperature for 48 hours without agitation.
  • All control and stressed samples were colorless and slightly opalescence without product related particulate. There was no observed difference in the degree of opalescence between the control samples and the freeze/thaw and agitation samples or the different levels of PS-80.
  • the protein concentration was also assessed for controls and stressed formulations. The protein concentration was between 19 and 22 mg/mL (Table 22). No significant changes or trends were observed upon freeze/thaw or agitation stress.
  • the DLS analysis showed that all samples had a hydrodynamic radius between 4.1 and 4.5 with a percent polydispersity between 10.7% and 19.4% (Table 23 and Table 24). No significant differences or trends were observed in the DLS relative to formulation or stress condition.
  • Table 25 Table 26 [0170] Sub-visible particulates were measured by HIAC for the T0, 1 freeze/thaw cycle, 3 freeze/thaw cycles, agitation control, 24 hour agitation, and 48 hour agitation samples with varying concentrations of PS-80. The results are summarized in Table 27 and Table 28. For all samples the cumulative counts/mL ranged from 2330 – 45270, 390 – 35857, 78 – 6430, and 0 – 150 for ⁇ 2 ⁇ m, 5 ⁇ m, ⁇ 10 ⁇ m, ⁇ 25 ⁇ m, respectively. A few conditions had elevated particulate levels relative to the other conditions of the same formulation, but these samples appear to be outliers rather than representing a trend. For example, 20 mM phosphate, 250 mM trehalose, 0.04% PS-80, pH 7.0 solution had 2842 ⁇ 10 ⁇ m particles at the initial timepoint, but only 155 particles in the agitation control.
  • Table 27 Table 28 [0171] IEX-HPLC was performed for all the 20 mM phosphate formulations at all conditions.
  • the main peak percentage for the T0 control at pH 7.0 with 0%, 0.02%, 0.04%, and 0.06% PS-80 was 68.88%, 69.83%, 71.36%, and 70.38%, respectively (Table 29).
  • the main peak percentage for the T0 control at pH 6.5, 7.0, and 7.5 was 70.11%, 71.36%, and 68.04%, respectively.
  • Example 4 Salt/Trehalose Dependence Study [0172] The preformation salt/trehalose dependence study performed at 22 mg/mL L-asparaginase was formulated in nine different formulation buffers, according to Table 30. An aliquot of 27 mL of Lot RE-LAP-P59 was divided into 6 portions of approximately 3 mL (3 portions) and 6 mL (3 portions). A buffer exchange was performed for formulations 1-6 via dialysis using 10K MWCO Slide-A-Lyzer G2 Cassettes. Dialysis was performed at room temperature at a minimum buffer to sample volume ratio of 40:1. Four dialysis buffer exchanges were performed for each formulation approximately every 2 hours, with one exchange occurring overnight.
  • Table 30 [0173] Following dialysis, the material was removed from the dialysis cassettes and the pH and protein concentration were confirmed. The concentration of the samples was then adjusted to 20 ⁇ 2 mg/mL with the corresponding buffer. After adjustment, the material in buffers 1-3 was split into equal portions of 3mL each. One set of these aliquots was supplemented with 10% PS-80 to a final concentration of 0.02%. All samples were then filtered and vialed in a biosafety cabinet. One set of the formulations were stored at 5°C until testing for the initial material quality evaluation, one set was stored at 5°C for 3 weeks, and one set was subjected to a heat stress at 40°C for 3 weeks.
  • the 3 week time point was selected after an evaluation of the visual appearance for after 1.5 weeks storage at 40°C. Formulations one and two at 5°C were additionally tested by IEX and SEC HPLC after seven weeks.
  • the salt/trehalose dependence study was performed to assess the impact of sodium chloride concentration on L-asparaginase formulations in 20 mM or 50 mM phosphate, 175 mM trehalose, at pH 7.0 with and without 0.02% PS-80 (Table 30). The formulations were exposed to 1.5 and 3 weeks at 5°C and 40°C. Size exclusion and IEX chromatography were used to assess the stability of RC in the presence of these varying salt concentrations in the selected buffers.
  • Osmolality and conductivity were also determined at the initial timepoint and are shown in Table 31.
  • Table 31 [0175] By appearance testing, all initial and stressed samples were colorless and slightly opalescence with very-few to no product related particulates. All formulations became more opalescent as a function of stress. The 0.02% PS-80 containing formulations did not have any particulates present regardless of storage duration (Table 32). This result indicates that addition of 0.02% PS-80 may delay particle formulation during long term storage. There was no dependence of appearance on salt concentration. The protein concentration was also assessed for initial and stressed formulations. The protein concentration was between 19.7 and 25.2 mg/mL (Table 33). No significant changes or trends were observed in the stressed samples.
  • Formulations with salt have slightly higher main peak percentages, compared to formulations without salt. Addition of 0.02% PS-80 had little to no benefit in terms of charge heterogeneity.
  • Table 34 [0177] SEC-HPLC results at the initial time point showed similar profiles with main peak percentages between 98.0% and 98.2% and HMW peak percentages between 1.9% and 2.0%. After three weeks at 40°C the samples showed slight degradation in purity due to the appearance of a LMW peak. The main peak percentages for the three week stressed samples were between 94.8% and 95.4%, with HMW peak percentages between 1.9% and 2.1% and LMW peak percentages between 2.7% and 3.1%. After three weeks at 5°C no significant changes in main peak and HMW peak percentages were observed (Table 35).
  • SE-HPLC separation combined with MALLS performed on both RC-P and Erwinase identified the SEC main content as tetramer, with a molecular weight in the range of 133-134 kDa. Both materials showed dominant tetramer content.
  • HMW1 species for both materials was identified as the octamer with a MW in the range of 277-286 kDa, which was close to the theoretical octamer mass of 280 kDa.
  • HMW2 species observed only for Erwinase was identified as the hexadecamer (16-mer) with a MW of 554 kDa, which matched to the theoretical hexadecamer mass of 560 kDa.
  • iCIEF Results The iCIEF method is an orthogonal method to evaluate the charge distribution of RC-P with an applied electrical field. The overlays of the oxidized sample and the reference standard were analyzed. Their relative peak intensities are listed in Table 44. Both oxidized samples showed an increased acidic peak at pI around 8.5. Oxidation have no impact for the basic variants. Table 44: iCIEF Results for the Oxidized Sample and RS Low pH study: [0188] Both RC-P and Erwinase samples were subjected to low pH treatment with 50mM sodium phosphate at pH 3.4 at room temperature, then frozen at -80°C.
  • SE-UHPLC Results The SE-UHPLC method is used to evaluate the size distribution of RC-P in its native state under nondenaturing conditions.
  • iCIEF Results The iCIEF method is an orthogonal method to evaluate the charge distribution of RC-P with an applied electrical field. The overlays of the low pH treated RC-P, Erwinase and the reference standard were analyzed. Their relative peak intensities are listed in Table 47. Both low pH treated RC-P and Erwinase showed an increased acidic peak at pI around 7.2, which was identified as monomer previously. Low pH treatment had no impact for the basic variants.
  • Erwinase has glycation modifications, which contribute to the acidic variants differences.
  • the higher level of octamer/16-mer observed in Erwinase contributed to the late eluting basic variants shown on CIEX.
  • Previous forced degradation study of RC-P demonstrates the glycated RC-P have comparable structure and activity to RC-P. Hydrophobic profiles, as measured by HIC and RP-UHPLC, are comparable for both materials. Minor differences were observed are due to the glycation modification and higher level of HMW content of Erwinase. Primary structure and higher order structure of both materials are comparable. Potency results are comparable.
  • Embodiment 1 An aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 °C for one week.
  • LMW low-molecular-weight
  • Embodiment 2 The formulation of embodiment 1, wherein the L- asparaginase is present at a concentration of about 20 mg/mL.
  • Embodiment 3 The formulation of embodiment 1 or embodiment 2, wherein the L-asparaginase is non-PEGylated and non-PASylated.
  • Embodiment 4 The formulation of any one of embodiments 1-3, wherein the one or more disaccharides comprise trehalose.
  • Embodiment 5 The formulation of embodiment 4, wherein the trehalose is present at a concentration of between about 50 mM and about 300 mM.
  • Embodiment 6 The formulation of embodiment 4, wherein the trehalose is present at a concentration of between about 150 mM and about 275 mM.
  • Embodiment 7 The formulation of embodiment 4, wherein the trehalose is present a concentration of about 170 mM.
  • Embodiment 8 The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise a phosphate buffer, an acetate buffer, or any combination thereof.
  • Embodiment 9 The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise a phosphate buffer.
  • Embodiment 10 The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise sodium phosphate.
  • Embodiment 11 The formulation of embodiment 10, wherein the sodium phosphate is present at a concentration of between about 0.5 mM and about 50 mM.
  • Embodiment 12 The formulation of embodiment 10, wherein the sodium phosphate is present at a concentration of about 20 mM.
  • Embodiment 13 The formulation of any one of embodiments 1-12, wherein the formulation further comprises sodium chloride.
  • Embodiment 14 The formulation of embodiment 13, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM.
  • Embodiment 15 The formulation of embodiment 13, wherein the sodium chloride is present at a concentration of about 50 mM.
  • Embodiment 16 The formulation of any one of embodiments 1-15, wherein the formulation further comprises one or more excipients.
  • Embodiment 17 The formulation of embodiment 16, wherein the one or more excipients comprise polysorbate 80.
  • Embodiment 18 The formulation of embodiment 17, wherein the polysorbate 80 is present a concentration of between about 0.004% (w/v) and about 0.2% (w/v).
  • Embodiment 19 The formulation of embodiment 17, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v).
  • Embodiment 20 The formulation of any one of embodiments 1-19, wherein the formulation has a pH of between about 4.0 and about 8.5.
  • Embodiment 21 The formulation of any one of embodiments 1-19, wherein the formulation has a pH of about 7.0.
  • Embodiment 22 A method of treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject a formulation of any one of embodiments 1-21.
  • Embodiment 23 The method of embodiment 22, wherein the disease, condition, or disorder is cancer.
  • Embodiment 24 The method of embodiment 23, wherein the cancer is acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • Embodiment 25 The method of embodiment 24, wherein the ALL is relapsed ALL.
  • Embodiment 26 The method of embodiment 23, wherein the cancer is lymphoblastic lymphoma (LBL).
  • Embodiment 27 The method of embodiment 26, wherein the LBL is relapsed LBL.
  • Embodiment 28 The method of any one of embodiments 22-27, wherein the formulation is administered intramuscularly.
  • Embodiment 29 The method of any one of embodiments 22-27, wherein the formulation is administered intravenously.
  • Embodiment 30 The method of any one of embodiments 22-29, wherein the formulation is co-administered with one or more other chemotherapeutic agents.
  • Embodiment 31 A kit, comprising: (i) a formulation of any one of embodiments 1-21; and (ii) instructions for treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof.
  • Embodiment 32 The kit of embodiment 31, wherein the disease, condition, or disorder is cancer.
  • Embodiment 33 The kit of embodiment 32, wherein the cancer is acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • Embodiment 34 The kit of embodiment 33, wherein the ALL is relapsed ALL.
  • Embodiment 35 The kit of embodiment 32, wherein the cancer is lymphoblastic lymphoma (LBL).
  • Embodiment 36 The kit of embodiment 35, wherein the LBL is relapsed LBL.

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