Preparation of L-asparaginase

Abstract

Provided herein are stable, aqueous, non-lyophilized formulations of L-asparaginase. Also provided herein are methods of using such formulations in the treatment of one or more diseases, disorders, or conditions treatable by asparagine depletion (e.g., cancers, such as acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL)).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 171,429, filed April 6, 2021, the entire disclosure of which is incorporated herein by reference.

[0002] Submission of sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 210463000300SEQLIST.TXT, Recorded: April 5, 2021, Size: 3KB). [Background technology]

[0003] 2. Background of the Invention Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginase, have been used successfully to treat a variety of potentially fatal diseases, including cancers such as acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), the majority of which affect children.

[0004] L-asparaginase of bacterial origin has high immunogenicity and antigenic potential. The first-line treatments on the market today are L-asparaginase and pegaspargase derived from E. coli. These products can induce adverse hypersensitivity reactions in patients, including allergic reactions, asymptomatic inactivation, and anaphylactic shock. Patients who experience hypersensitivity reactions to these products often need to discontinue treatment, resulting in poor prognosis and survival. These patients have turned to Erwinaze® after experiencing hypersensitivity reactions. Erwinaze® has suffered from long-standing supply issues, which reportedly can take up to nine 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). The problem continues to this day, and Erwinase® shortages are common. The FDA has issued administrative warning letters to manufacturers, stating that "alterations in source material or cell lines have a substantial potential to adversely affect the identity, potency, quality, purity, or efficacy of Erwinase®."

[0005] There is a need for therapeutic options that are free of immunogenic cross-reactivity. L-asparaginase (such as L-asparaginase recombinantly produced in Pseudomonas fluorescens) that is free of immunological cross-reactivity to E. coli derived asparaginase would address a significant medical need for ALL / lymphoblastic lymphoma (LBL) patients by helping to ensure the availability of asparaginase to patients who have developed hypersensitivity to E. coli derived asparaginase (as a component of a multi-agent chemotherapy regimen).

[0006] Currently, Erwinase® is supplied in vials as a sterile, lyophilized, white powder. 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). The availability of a non-lyophilized formulation would eliminate the need for rehydration prior to administration to a subject, e.g., a human. To this end, an alternative non-lyophilized treatment option, Oncaspar®, is supplied as a clear, colorless, preservative-free, mesotonic sterile solution in phosphate buffered saline, pH 7.3. Each milliliter contains 750±150 International Units of pegaspargase, disodium phosphate (USP) (5.58 mg), monosodium phosphate (USP) (1.20 mg), and sodium chloride (USP) (8.50 mg) in water for injection (USP). Yet another non-lyophilized alternative, ASPARLAS®, is supplied as a clear, colorless, preservative-free, mesotonic sterile solution in phosphate-buffered saline at pH 7.3 that requires dilution prior to intravenous infusion. Each vial of ASPARLAS® contains 3,750 units in a 5 mL solution. Each milliliter contains 750 units of calaspargase pegol-mknl, disodium phosphate (USP) (5.58 mg), monosodium phosphate (USP) (1.20 mg), and sodium chloride (USP) (8.50 mg) in water for injection (USP). However, although non-lyophilized formulations exist (e.g., Oncaspar® and ASPARLAS®), stability issues exist, for example, existing formulations must be used within 48 hours when stored at room temperature.

[0007] Thus, there is a need in the art for a stable, non-lyophilized, aqueous formulation of L-asparaginase. Summary of the Invention

[0008] Provided herein are aqueous, non-lyophilized formulations of L-asparaginase. In some aspects, such formulations may find use in treating one or more diseases, disorders, or conditions treatable by asparagine depletion, including cancers such as acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), including relapsed ALL and relapsed LBL.

[0009] In one aspect, an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence that is at least about 70% identical to SEQ ID NO:1; (ii) one or more stabilizers, or one or more buffers, or any combination thereof; Such formulations comprising:

[0010] In some embodiments, the one or more stabilizers include one or more disaccharides, one or more sorbitol, one or more amino acids, or any combination thereof. In some embodiments, the one or more disaccharides include trehalose, sucrose, or any combination thereof. In some embodiments, the one or more disaccharides include trehalose. In some embodiments, the one or more buffers are substantially free of amino acids. In some embodiments, the one or more buffers include a phosphate buffer, an acetate buffer, or any combination thereof. In some embodiments, the one or more buffers include a phosphate buffer. In some embodiments, the one or more buffers include sodium phosphate. In some embodiments, the sodium phosphate is disodium phosphate (anhydrous), monosodium phosphate monohydrate, or a combination thereof. In some embodiments, the formulation further includes sodium chloride. In some embodiments, the formulation further includes one or more excipients. In some embodiments, the one or more excipients include polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, 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.

[0011] In some embodiments, the formulation contains less than about 0.6% low molecular weight (LMW) species after storage for 2 months at 40° C. In some embodiments, the formulation contains less than about 0.6% low molecular weight (LMW) species after storage for 1 week at 37° C. In some embodiments, the formulation contains less than 2% high molecular weight (HMW) species after storage for 2 months at 40° C.

[0012] In one aspect, an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence that is at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides, including trehalose, sucrose, or any combination thereof; (iii) one or more buffers that are substantially free of amino acids; and containing less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week.

[0013] In another aspect, provided herein is a method of treating a disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof comprising administering to the subject any of the formulations described herein.

[0014] In another aspect, provided herein is a unit dosage form comprising (i) any of the formulations described herein, and (ii) one or more pharma- ceutically acceptable excipients.

[0015] In another aspect, provided herein is a kit comprising (i) any of the formulations described herein and (ii) instructions for treating a disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] definition "Subject" refers to a mammal, including 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, the subject refers to a human.

[0017] The articles "a" and "an" are used in this specification and the appended claims 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. As an example, "a buffer" means one buffer or more than one buffer.

[0018] As used herein, "about" a parameter or value includes and describes the parameter or value itself. For example, "about X" includes and describes X itself. As used herein, the term "about" to modify, for example, dimensions, volumes, amounts of components in a composition, concentrations, process temperatures, process times, yields, flow rates, pressures, and similar values ​​and ranges thereof, refers to variations in quantity that may occur, for example, through typical measuring and handling procedures used to make a compound, composition, concentrate, or formulation to be used, through unintentional errors in these procedures, through differences in the manufacture, source, or purity of starting materials or components used to carry out the method, and similar circumstances. The term "about" also encompasses amounts that differ from a particular initial concentration or mixture, for example, due to aging of a composition, formulation, or cell culture, and amounts that differ from a particular initial concentration or mixture due to mixing or processing of a composition or formulation. The claims appended hereto include equivalents to these amounts, whether modified by the term "about". The term "about" may also refer to a range of values ​​similar to the stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that is within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of the recited reference value.

[0019] As used herein, the terms "treatable by depletion of asparagine" and "treatable by asparagine depletion" refer to a disease, condition, or disorder in which cells involved in or causing the disease, condition, or disorder either do not have or have a reduced ability to synthesize L-asparagine. L-asparagine depletion or deficiency may be partial or substantially complete (e.g., at levels undetectable using methods or devices 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:1, but may include additional amino acids.

[0021] "Low molecular weight species" or "LMW species" as used herein refers to species having a molecular weight lower than that of L-asparaginase. In some embodiments, 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. In some embodiments, LMW species are formed by degradation or breakdown of L-asparaginase, for example, via hydrolysis of one or more covalent bonds in L-asparaginase.

[0022] "High molecular weight species" or "HMW species," as used herein, refers to species having a molecular weight higher than that of L-asparaginase. In some embodiments, the HMW species has 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.

[0023] "Trehalose" refers to a disaccharide having the following structure: TIFF2024515555000001.tif32128.

[0024] "Sucrose" refers to a disaccharide having the following structure: TIFF2024515555000002.tif47128.

[0025] "Polysorbate 80" or "PS-80" refers to an excipient having the following structure: TIFF2024515555000003.tif47132.

[0026] "PEGylated" as used herein 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 manner (e.g., via one or more covalent bonds) and at any suitable position of the structure or moiety in question. For example, "PEGylated L-asparaginase" refers to L-asparaginase to which one or more PEG units are conjugated or attached. Thus, a "non-PEGylated" moiety is one to which PEG is not conjugated or attached.

[0027] "PASylated" as used herein is used to describe a structure or moiety to which one or more polypeptide units containing proline, alanine, or serine, or any combination thereof, are conjugated or attached. Any suitable number of such polypeptide units may be attached in any suitable manner (e.g., via one or more covalent bonds) and at any suitable position of the structure or moiety in question. For example, "PASylated L-asparaginase" refers to L-asparaginase to which one or more polypeptide units containing proline, alanine, or serine, or any combination thereof, are conjugated or attached. Thus, a "non-PASylated" moiety is one to which one or more polypeptide units containing proline, alanine, or serine, or any combination thereof, are not conjugated or attached.

[0028] "SEQ ID NO:1" is as follows: ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSAR YITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY.

[0029] formulation In one aspect, provided herein is an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having 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.

[0030] In some embodiments, the L-asparaginase formulation comprises one or more stabilizers. In some embodiments, the one or more stabilizers comprise one or more disaccharides, one or more sorbitol, one or more amino acids, or any combination thereof. In some embodiments, 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 are substantially free of amino acids. 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 2 months. In some embodiments, the L-asparaginase formulation comprises less than 2% high molecular weight (HMW) species after storage at 40° C. for 2 months. In some embodiments, the L-asparaginase formulations contain less than about 0.6% low molecular weight (LMW) species and less than 2% high molecular weight (HMW) species after two months of storage at 40° C. In some embodiments, the L-asparaginase formulations contain less than about 0.6% low molecular weight (LMW) species after one week of storage at 37° C.

[0031] In one aspect, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; and (iii) one or more buffers substantially free of amino acids, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week.

[0032] In one aspect, the L-asparaginase according to the present disclosure provided herein is a recombinant L-asparaginase. In a further aspect, the L-asparaginase according to the present invention described herein is an enzyme having L-asparagine aminohydrolase activity. Such enzymatic activity of L-asparaginase may include deamidation of asparagine to aspartic acid and ammonia, as well as deamidation of glutamine to glutamic acid and ammonia.

[0033] In some embodiments, the L-asparaginase disclosed herein is active as a multimer. In some embodiments, the L-asparaginase is an active enzyme as a tetramer. The tetramer is composed of four subunits (also known as monomers). In some embodiments, the L-asparaginase is a tetramer consisting of four identical 35 kD subunits. In some embodiments, the L-asparaginase is a non-disulfide-linked tetrameric therapeutic protein. In certain embodiments, each of the subunits or monomers of the multimeric L-asparaginase comprises the amino acid sequence of SEQ ID NO:1.

[0034] In certain embodiments, each of the subunits or monomers of the tetrameric L-asparaginase comprises the amino acid sequence of SEQ ID NO: 1. In another embodiment, the L-asparaginase is derived from Erwinia chrysanthemi NCPPB1066 (Genbank Accession No. CAA32884, incorporated herein by reference in its entirety), either with or without a signal peptide and / or leader sequence.

[0035] In some embodiments, the L-asparaginase is composed of multiple subunits, for example, four subunits or monomers (tetramer). The corresponding modified protein can then be composed of, for example, 1 to 20 (or more) peptides conjugated to each of the monomers of the tetramer. In some embodiments, 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 monomers of the L-asparaginase. In certain embodiments, the L-asparaginase is a multimer comprising multiple subunits or monomers, for example, a tetramer, in which each of the monomers in the tetramer is conjugated to one peptide, resulting in a tetramer comprising four conjugated peptides, one for each monomer. In some embodiments, the L-asparaginase is a tetramer that contains 1 to 4 peptides conjugated to each of the monomers.

[0036] In some embodiments, the L-asparaginase is a tetramer that includes 4-20 peptides conjugated to each of the L-monomers. In some embodiments, the L-asparaginase is a tetramer that includes 6-18 peptides conjugated to each of the monomers. In some embodiments, the L-asparaginase is a tetramer that includes 6-18 peptides conjugated to each of the monomers. In some embodiments, the L-asparaginase is a tetramer that includes 10-15 peptides conjugated to each of the monomers.

[0037] In one aspect, the present invention relates to a modified protein comprising an L-asparaginase and a plurality of chemically bonded peptide sequences, hi further aspects, the length of the peptide sequences is about 10 to about 100, about 15 to about 60, or about 20 to about 40.

[0038] Fragments of L-asparaginase, preferably a fragment of the L-asparaginase of SEQ ID NO: 1, may be useful in the invention described herein. The term "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 contain fewer amino acids than the L-asparaginase exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1), but sufficient amino acids to confer L-aminohydrolase activity. For example, a "fragment of L-asparaginase" is a fragment of at least about 150 or 200 consecutive amino acids (e.g., 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 consecutive amino acids) of one of the L-asparaginases exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1), and / or the above fragment is a fragment of at most 50 (e.g., at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, or 326 consecutive amino acids) from the N-terminus of the above L-asparaginase exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1). 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids have been deleted from the C-terminus of the L-asparaginase exemplified herein (e.g., the L-asparaginase of SEQ ID NO:1), and / or up to 75 or 100 amino acids (e.g., 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 amino acids) have been deleted from the C-terminus of the L-asparaginase exemplified herein (e.g., the L-asparaginase of SEQ ID NO:1), and / or amino acids have been deleted at both the N-terminus and the C-terminus of the L-asparaginase exemplified herein (e.g., the L-asparaginase of SEQ ID NO:1), where the total number of deleted amino acids may be up to 125 or 150 amino acids.

[0039] Indeed, those skilled in the art will understand how to select and design homologous proteins that substantially retain their L-asparaginase activity. Typically, the Nessler assay is used to determine L-asparaginase activity according to the method described by Mashburn and Wriston (Mashburn, L., and Wriston, J. (1963) "Tumor Inhibitory Effect of L Asparaginase," Biochem Biophys Res Commun 12, 50 (incorporated herein by reference in its entirety)).

[0040] It is well known in the art that a polypeptide can be modified by substitution, insertion, deletion, and / or addition of one or more amino acids while retaining its enzymatic activity. In this context, the term "one or more amino acids" can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. For example, the substitution of one amino acid at a given position with a chemically equivalent amino acid that does not affect the functional properties of the protein is a common substitution that can be defined as an exchange in 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.

[0041] Thus, changes that result in the substitution of one uncharged residue for another (e.g., aspartic acid for glutamic acid), or one positively charged residue for another (e.g., arginine for lysine), would be expected to produce functionally equivalent products.

[0042] The position where the amino acid is modified and the number of amino acids that can be modified in the amino acid sequence are not particularly limited. Those skilled in the art can recognize modifications that can be introduced without affecting the activity of the protein. For example, modifications at the N- or C-terminal part of the protein can be expected not to change the activity of the protein under certain circumstances. In particular, for asparaginase, much characterization needs to be done, especially regarding the sequence, structure, and residues that form the active catalytic site. This provides guidance regarding the residues that can be modified without affecting the activity of the enzyme. All L-asparaginases from known bacterial sources share common structural characteristics. All are homotetramers with four active sites between the N- and C-terminal domains of two adjacent monomers (Aghaipour (2001) Biochemistry 40, 5655-5664, incorporated herein by reference in its entirety). All have a high degree of similarity in their tertiary and quaternary structures (Papageorgiou (2008) FEBS J. 275, 4306-4316, incorporated herein by reference in its entirety). The sequence of the catalytic site of L-asparaginase is highly conserved between Erwinia chrysanthemi, Erwinia carotovora, and E. coli L-asparaginase II (ibid.). The mobile loop of the active site contains amino acid residues 14-33, and structural analysis indicates that Thrl5, Thr95, Ser62, Glu63, Asp96, and Alal20 contact the ligand (ibid.). Aghaipour et al. carried out a detailed analysis of the four active sites of Erwinia chrysanthemi L-asparaginase by examining the high-resolution crystal structure of the enzyme complexed with its substrate (Aghaipour (2001) Biochemistry 40, 5655-5664).Kotzia et al. provide sequences of L-asparaginase from several Erwinia species and subspecies, and although the proteins have only about 75-77% identity between Erwinia chrysanthemi and Erwinia carotovora, each of these still has L-asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657-669). Moola et al. performed epitope mapping studies of Erwinia chrysanthemi 3937 L-asparaginase, and found that even after mutating various antigenic sequences in an attempt to reduce the immunogenicity of the asparaginase, these were able to retain enzymatic activity (Moola (1994) Biochem. J. 302, 921-927). Given the extensive characterization that has been performed on L-asparaginase, it is possible for one skilled in the art to determine how to make fragments and / or sequence substitutions while still retaining enzymatic activity.

[0043] More specifically, the definition of a protein used for the L-asparaginase of the present invention also includes fragments of the protein of SEQ ID NO: 1. The term "fragment of SEQ ID NO: 1" means that the polypeptide sequence may contain fewer amino acids than the full-length SEQ ID NO: 1, but retains enough of the protein to confer L-aminohydrolase activity. In some embodiments, the L-asparaginase has at least about 80% homology or identity to a protein comprising SEQ ID NO: 1.

[0044] In some embodiments, the L-asparaginase of the formulation comprises four monomeric units, each monomeric unit having an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1. In some embodiments, each monomeric unit has an amino acid sequence 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. In some embodiments, each monomeric unit has an amino acid sequence 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.

[0045] In some embodiments, the L-asparaginase of the formulation is present at a concentration of about 20 mg / mL.

[0046] In some embodiments, the L-asparaginase in the formulation is non-PEGylated. In some embodiments, the L-asparaginase in the formulation is non-PASylated. In some embodiments, the L-asparaginase in the formulation is non-PEGylated and non-PASylated.

[0047] In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation comprises one or more stabilizers. In some embodiments, in conjunction with the above or below embodiments, the one or more stabilizers comprise one or more disaccharides, one or more sorbitols, one or more amino acids, or any combination thereof. In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation comprises one or more buffers. In some embodiments, in conjunction with the above or below embodiments, the one or more buffers comprise acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane (e.g., Tris), or any combination thereof. In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation comprises one or more surfactants. In some embodiments, in conjunction with the above or below embodiments, the one or more surfactants comprise polysorbate 80 (e.g., Tween 80), polysorbate 20 (e.g., Tween 20), poloxamer 188, or any combination thereof. In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation includes one or more polyols (e.g., mannitol and sorbitol), one or more disaccharides (e.g., sucrose and trehalose), and one or more polysaccharides (e.g., dextran 40). In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation includes sodium chloride. In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation includes one or more amino acids (e.g., proline, glycine, and arginie).

[0048] In some embodiments, in conjunction with any of the above or below embodiments, the formulation comprises one or more stabilizers at a concentration of about 50 mM to about 300 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 75 mM to about 300 mM, about 100 mM to about 300 mM, about 125 mM to about 300 mM, about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300 mM, or about 275 mM to about 300 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 50 mM to about 275 mM, about 50 mM to about 250 mM, about 50 mM to about 225 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, or about 50 mM to about 75 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 150 mM to about 275 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 150 mM to about 200 mM, about 150 mM to about 190 mM, about 150 mM to about 180 mM, 150 mM to about 170 mM, or about 160 mM to 150 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 160 mM to about 200 mM, about 170 mM to about 200 mM, about 180 mM to about 200 mM, or about 190 mM to about 200 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 170 mM.

[0049] In some embodiments, 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 about 50 mM to about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 75 mM to about 300 mM, about 100 mM to about 300 mM, about 125 mM to about 300 mM, about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300 mM, or about 275 mM to about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 50 mM to about 275 mM, about 50 mM to about 250 mM, about 50 mM to about 225 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, or about 50 mM to about 75 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 150 mM to about 275 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 150 mM to about 200 mM, about 150 mM to about 190 mM, about 150 mM to about 180 mM, 150 mM to about 170 mM, or about 160 mM to 150 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 160 mM to about 200 mM, about 170 mM to about 200 mM, about 180 mM to about 200 mM, or about 190 mM to about 200 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 170 mM.

[0051] In some embodiments, the formulation comprises trehalose at a concentration of about 50 mM to about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 75 mM to about 300 mM, about 100 mM to about 300 mM, about 125 mM to about 300 mM, about 150 mM to about 300 mM, about 175 mM to about 300 mM, about 200 mM to about 300 mM, about 225 mM to about 300 mM, about 250 mM to about 300 mM, or about 275 mM to about 300 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 50 mM to about 275 mM, about 50 mM to about 250 mM, about 50 mM to about 225 mM, about 50 mM to about 200 mM, about 50 mM to about 175 mM, about 50 mM to about 150 mM, about 50 mM to about 125 mM, about 50 mM to about 100 mM, or about 50 mM to about 75 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 150 mM to about 275 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 150 mM to about 200 mM, about 150 mM to about 190 mM, about 150 mM to about 180 mM, 150 mM to about 170 mM, or about 160 mM to 150 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 160 mM to about 200 mM, about 170 mM to about 200 mM, about 180 mM to about 200 mM, or about 190 mM to about 200 mM. In some embodiments, the formulation comprises trehalose at a concentration of about 170 mM.

[0052] In some embodiments, one or more buffers of the formulation are substantially free of amino acids. In some embodiments, one or more buffers of the formulation are substantially free of histidine or arginine, or both. In some embodiments, one or more buffers of the formulation are substantially free of histidine. In some embodiments, one or more buffers of the formulation comprise a phosphate buffer, an acetate buffer, or any combination thereof. In some embodiments, one or more buffers comprise a phosphate buffer. In some embodiments, one or more buffers comprise an acetate buffer. In some embodiments, one or more buffers comprise a combination of a phosphate buffer and an acetate buffer. In some embodiments, one or more buffers comprise an alkali metal acetate. In some embodiments, one or more buffers comprise an alkali metal phosphate. In some embodiments, one or more buffers comprise sodium phosphate.

[0053] In some embodiments, the formulation comprises one or more buffers at a concentration of about 0.5 mM to about 50 mM. In some embodiments, the formulation comprises one or more buffers at a concentration of about 1 mM to about 50 mM, about 2.5 mM to about 50 mM, about 5 mM to about 50 mM, about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 50 mM, about 40 mM to about 50 mM, or about 45 mM to about 50 mM. In some embodiments, the formulation comprises one or more buffers at a concentration of about 0.5 mM to about 45 mM, about 0.5 mM to about 40 mM, about 0.5 mM to about 35 mM, about 0.5 mM to about 30 mM, about 0.5 mM to about 25 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 M to about 2.5 mM, or 0.5 mM to about 1 mM. In some embodiments, the formulation comprises one or more buffers at a concentration of about 10 mM to about 30 mM. In some embodiments, the formulation comprises one or more buffers at a concentration of about 15 mM to about 25 mM. In some embodiments, the 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 about 0.5 mM to about 50 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 1 mM to about 50 mM, about 2.5 mM to about 50 mM, about 5 mM to about 50 mM, about 10 mM to about 50 mM, about 15 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, about 30 mM to about 50 mM, about 35 mM to about 50 mM, about 40 mM to about 50 mM, or about 45 mM to about 50 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 0.5 mM to about 45 mM, about 0.5 mM to about 40 mM, about 0.5 mM to about 35 mM, about 0.5 mM to about 30 mM, about 0.5 mM to about 25 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 15 mM, about 0.5 mM to about 10 mM, about 0.5 mM to about 5 mM, about 0.5 M to about 2.5 mM, or 0.5 mM to about 1 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 10 mM to about 30 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 15 mM to 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 about 25 mM to about 150 mM. In some embodiments, the formulation further comprises sodium chloride at a concentration of about 30 mM to about 150 mM, about 40 mM to about 150 mM, about 50 mM to about 150 mM, about 60 mM to about 150 mM, about 70 mM to about 150 mM, about 80 mM to about 150 mM, about 90 mM to about 150 mM, about 100 mM to about 150 mM, about 110 mM to about 150 mM, about 120 mM to about 150 mM, about 130 mM to about 150 mM, or about 140 mM to about 150 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of about 25 mM to about 140 mM, about 25 mM to about 130 mM, about 25 mM to about 120 mM, about 25 mM to about 110 mM, about 25 mM to about 100 mM, about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, about 25 mM to about 50 mM, about 25 mM to about 40 mM, or about 25 mM to about 30 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of about 30 mM to about 70 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of about 40 mM to 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. In some embodiments, 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 about 0.3% (w / v) or less. In some embodiments, the formulation comprises one or more excipients at a concentration of about 0.28% (w / v) or less. In some embodiments, the formulation comprises one or more excipients at a concentration of about 0.26% (w / v) or less. In some embodiments, the formulation comprises one or more excipients at a concentration of about 0.24% (w / v) or less. In some embodiments, the formulation comprises one or more excipients at a concentration of about 0.22% (w / v) or less. In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.004% (w / v) to about 0.3% (w / v). In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.01% (w / v) to about 0.3% (w / v).

[0058] In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.004% (w / v) to about 0.2% (w / v). In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.004% (w / v) to about 0.18% (w / v), about 0.004% (w / v) to about 0.16% (w / v), about 0.004% (w / v) to about 0.14% (w / v), about 0.004% (w / v) to about 0.12% (w / v), about 0.004% (w / v) to about 0.1% (w / v), about 0.004% (w / v) to about 0.08% (w / v), about 0. In some embodiments, the compound is further contained at a concentration of about 0.004% (w / v) to about 0.06% (w / v), about 0.004% (w / v) to about 0.04% (w / v), about 0.004% (w / v) to about 0.03%, about 0.004% (w / v) to about 0.025% (w / v), about 0.004% (w / v) to about 0.02% (w / v), about 0.004% (w / v) to about 0.015% (w / v), or about 0.004% (w / v) to about 0.01% (w / v). In some embodiments, the formulation contains one or more excipients at about 0.01% (w / v) to about 0.2% (w / v), about 0.015% (w / v) to about 0.2% (w / v), about 0.02% (w / v) to about 0.2% (w / v), about 0.025% (w / v) to about 0.2% (w / v), about 0.03% (w / v) to about 0.2% (w / v), about 0.04% (w / v) to about 0.2% (w / v), about 0.05% (w / v) to about 0.2% (w / v), about 0.06% (w / v) to about In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.01% (w / v) to about 0.03% (w / v). In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.015% (w / v) to 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 about 0.004% (w / v) to about 0.2% (w / v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.004% (w / v) to about 0.18% (w / v), about 0.004% (w / v) to about 0.16% (w / v), about 0.004% (w / v) to about 0.14% (w / v), about 0.004% (w / v) to about 0.12% (w / v), about 0.004% (w / v) to about 0.1% (w / v), about 0.004% (w / v) to about 0.08% (w / v), about 0. In some embodiments, the compound is further contained at a concentration of about 0.004% (w / v) to about 0.06% (w / v), about 0.004% (w / v) to about 0.04% (w / v), about 0.004% (w / v) to about 0.03%, about 0.004% (w / v) to about 0.025% (w / v), about 0.004% (w / v) to about 0.02% (w / v), about 0.004% (w / v) to about 0.015% (w / v), or about 0.004% (w / v) to about 0.01% (w / v). In some embodiments, the formulation contains polysorbate 80 at about 0.01% (w / v) to about 0.2% (w / v), about 0.015% (w / v) to about 0.2% (w / v), about 0.02% (w / v) to about 0.2% (w / v), about 0.025% (w / v) to about 0.2% (w / v), about 0.03% (w / v) to about 0.2% (w / v), about 0.04% (w / v) to about 0.2% (w / v), about 0.05% (w / v) to about 0.2% (w / v), about 0.06% (w / v) to about 0.2% (w / v), about 0.08% (w / v) to about 0.2% (w / v), about 0.09% (w / v) to about 0.2% (w / v), about 0.10% (w / v) to about 0.2% (w / v), about 0.15 ... In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.01% (w / v) to about 0.03% (w / v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.015% (w / v) to 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 formulations provided herein have a pH of about 4.0 to about 8.5. In some embodiments, the formulations have a pH of about 4.5 to about 8.5, about 5.0 to about 8.5, about 5.5 to about 8.5, about 6.0 to about 8.5, about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.5 to about 8.5, or about 8.0 to about 8.5. In some embodiments, the formulations have a pH of about 4.5 to about 8.0, about 4.5 to about 7.5, about 4.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, or about 4.5 to about 5.0. In some embodiments, the formulations provided herein have a pH of about 4.0 to about 8.0, about 4.0 to about 7.5, about 4.0 to about 7.0, about 4.0 to about 6.5, about 4.0 to about 6.0, about 4.0 to about 5.5, about 4.0 to about 5.0, or about 4.0 to about 4.5. In some embodiments, the formulations provided herein have a pH of about 5.5 to about 8.5. In some embodiments, the formulations provided herein have a pH of about 6.0 to about 8.0. In some embodiments, the formulations provided herein have a pH of 6.5 to about 7.5. In some embodiments, the formulations provided herein have a pH of around 7.0.

[0061] In some embodiments, provided herein, in conjunction with the above or below embodiments, is an aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence 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. In some embodiments, the L-asparaginase formulation comprises one or more stabilizers. In some embodiments, the one or more stabilizers comprise one or more disaccharides, one or more sorbitol, one or more amino acids, or any combination thereof. In some embodiments, 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 are substantially free of amino acids. In some embodiments, the L-asparaginase formulation contains less than about 0.6% low molecular weight (LMW) species after storage for 2 months at 40° C. In some embodiments, the L-asparaginase formulation contains less than 2% high molecular weight (HMW) species after storage for 2 months at 40° C. In some embodiments, the L-asparaginase formulation contains less than about 0.6% low molecular weight (LMW) species and less than 2% high molecular weight (HMW) species after storage for 2 months at 40° C. In some embodiments, the L-asparaginase formulation contains less than about 0.6% low molecular weight (LMW) species and less than 2% high molecular weight (HMW) species after storage for 2 months at 40° C. In some embodiments, the L-asparaginase formulation contains less than about 0.6% low molecular weight (LMW) species after storage for 1 week at 37° C. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0062] In some embodiments, in conjunction with any of the above or below embodiments, (i) the L-asparaginase comprises four monomeric units, each monomeric unit having 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., a disaccharide, sorbitol, an amino acid, or any combination thereof) at a concentration of about 50 mM to about 300 mM; (iii) one or more buffers (e.g., acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane, or any combination thereof) at a concentration of about 0.5 mM to about 50 mM; (iv) sodium chloride present at a concentration of about 25 mM to about 150 mM; and (v) one or more surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof) at a concentration of about 0.004% (w / v) to about 0.3% (w / v). In some embodiments, in conjunction with the above or below embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, in conjunction with the above or below embodiments, the formulation has a pH of about 7.0.

[0063] In some embodiments, in conjunction with the above or below embodiments, (i) L-asparaginase comprises four monomeric units, each monomeric unit having an amino acid sequence at least about 70% identical to SEQ ID NO:1, and is present at a concentration of around 20 mg / mL; (ii) trehalose present at a concentration of about 50 mM to about 300 mM; (iii) sodium phosphate (e.g., disodium phosphate (anhydrous), sodium phosphate monohydrate, or a combination thereof) present at a concentration of about 0.5 mM to about 50 mM; (iv) sodium chloride present at a concentration of about 25 mM to about 150 mM; and (v) polysorbate 80 present at a concentration of about 0.004% (w / v) to about 0.2% (w / v).

[0064] In some embodiments, in conjunction with the above or below embodiments, L-asparaginase may be formulated in a unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary doses for human subjects and other mammals, each unit containing a predetermined amount of active ingredient calculated to produce a desired therapeutic effect in association with a suitable pharmaceutical excipient. In some embodiments, the unit dosage may be administered every 12 hours, every 24 hours, every 48 hours, or every 72 hours. In some embodiments, the unit dosage is administered every 48 hours. In some embodiments, the unit dose contains 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 L-asparaginase.

[0065] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; (iii) one or more buffers substantially free of amino acids; and (iv) sodium chloride, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0066] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; (iii) one or more buffers substantially free of amino acids; and (iv) one or more excipients, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week.

[0067] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; (iii) one or more buffers substantially free of amino acids; (iv) sodium chloride; and (v) one or more excipients, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0068] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomer units, each monomer unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; (iii) one or more buffers substantially free of amino acids; and (iv) polysorbate 80, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0069] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomer units, each monomer unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; (iii) one or more buffers substantially free of amino acids; (iv) sodium chloride; and (v) polysorbate 80, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0070] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase comprising four monomer units, each monomer unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) trehalose; (iii) sodium phosphate; (iv) sodium chloride; and (v) polysorbate 80, the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0071] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase, the L-asparaginase comprising four monomer units, each monomer unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1, the L-asparaginase being present at a concentration of around 20 mg / mL; (ii) trehalose present at a concentration of about 50 mM to about 300 mM; (iii) sodium phosphate present at a concentration of about 0.5 mM to about 50 mM; (iv) sodium chloride present at a concentration of about 25 mM to about 150 mM; and (v) polysorbate 80 present at a concentration of about 0.004% (w / v) to about 0.2% (w / v); the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5, hi some embodiments, the formulation has a pH of about 7.0.

[0072] In some embodiments, provided herein is an aqueous, non-lyophilized formulation comprising: (i) L-asparaginase, the L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1, the L-asparaginase being present at a concentration of about 20 mg / mL; (ii) trehalose present at a concentration of about 170 mM; (iii) sodium phosphate present at a concentration of about 20 mM; (iv) sodium chloride present at a concentration of about 50 mM; and (v) polysorbate 80 present at a concentration of about 0.02% (w / v); the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0073] In some embodiments, provided herein is a formulation as described elsewhere herein, wherein the formulation comprises less than about 5% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 4.5% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 4% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 3.5% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 3% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 2.5% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulation comprises less than about 2% low molecular weight (LMW) species after 1 week storage at 37° C. The formulations contain less than about 1.5% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulations contain less than about 1% low molecular weight (LMW) species after 1 week storage at 37° C. In some embodiments, the formulations contain less than about 0.5% low molecular weight (LMW) species after 1 week storage at 37° C.

[0074] It is to be understood that each component and / or feature of the formulations described herein may be present in combination with any other component and / or feature of the formulations described herein, just as if each and every combination of the components and / or features described herein were individually and specifically recited.

[0075] Treatment method The L-asparaginase formulations of the present disclosure can be used to treat a disease treatable by asparagine depletion or administration of asparaginase in a subject (e.g., a human). In some embodiments, the human subject experiences asymptomatic inactivation of E. coli derived asparaginase prior to administration of L-asparaginase. In some embodiments, the subject experiences an allergic reaction to E. coli derived asparaginase prior to administration of L-asparaginase. In some embodiments, the subject experiences anaphylaxis to E. coli derived asparaginase prior to administration of L-asparaginase. Non-limiting examples of objective signs of allergy or hypersensitivity include testing "antibody positive" to the asparaginase enzyme.

[0076] In some embodiments, the L-asparaginase formulations of the present disclosure are useful for treating, or manufacturing a medicament for use in treating, acute lymphoblastic leukemia (ALL). Relapse rates in ALL patients after treatment with L-asparaginase remain high, with approximately 10-25% of pediatric ALL patients experiencing early relapse (e.g., some during the maintenance phase at 30-36 months after induction). If a patient treated with E. coli-derived L-asparaginase experiences a relapse, subsequent treatment with E. coli preparations can result in a "vaccination" effect, whereby the E. coli preparation becomes increasingly immunogenic during subsequent administrations. In one embodiment, the L-asparaginase of the present invention can be used to treat relapsed ALL patients previously treated with other asparaginase preparations, particularly relapsed ALL patients previously treated with E. coli-derived asparaginase.

[0077] Diseases or disorders for which the L-asparaginase formulations of the present disclosure are useful for treating include, but are not limited to, malignancies or cancers, including, but not limited to, hematological malignancies, lymphoma, non-Hodgkin's lymphoma, NK lymphoma, pancreatic cancer, Hodgkin's disease, large cell immunoblastic lymphoma, acute promyelocytic leukemia, acute myeloid leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), mixed B-cell myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum cell sarcoma, and melanosarcoma, and diffuse large B-cell lymphoma (DLBCL). Other diseases or disorders that L-asparaginase preparations are useful for treating include, but are not limited to, cancers such as renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforme and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung cancer including large cell lung carcinoma and small cell lung carcinoma, endometrial cancer, ovarian adenocarcinoma, ovarian teratocarcinoma, cervical adenocarcinoma, breast cancer, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon cancer, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate cancer, osteosarcoma, bone epithelioid carcinoma, prostate cancer, and thyroid cancer.Cancer can be a solid cancer, such as lung cancer or breast cancer.Exemplary non-malignant hematological diseases that respond to asparagine depletion include immune system-mediated hematological diseases, including, but not limited to, infectious diseases such as those caused by HIV infection (i.e., AIDS). Non-hematological diseases associated with asparagine dependency include autoimmune diseases such as rheumatoid arthritis, collagen vascular disease, AIDS, osteoarthritis, Isaac'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 biliary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease. Cells suspected of causing the disease may be tested for asparagine dependency in any suitable in vitro or in vivo assay, for example, an in vitro assay in which the growth medium lacks asparagine.

[0078] In some embodiments, in conjunction with the above or below embodiments, the disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof comprises WNT-mutated colorectal cancer (CRC).In some embodiments, in conjunction with the above or below embodiments, the disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof comprises relapsing-remitting (R / R) acute myeloid leukemia (AML).

[0079] Diseases or disorders that the L-asparaginase formulations of the present disclosure are useful for treating include, but are not limited to, sarcoma, breast cancer, metastatic breast cancer, liver cancer, gastric cancer, colorectal cancer, and head and neck cancer.

[0080] Pharmaceutical Compositions and Co-Administration In some embodiments, the L-asparaginase formulations of the present disclosure are co-administered with a multi-agent chemotherapy regimen. In some embodiments, the L-asparaginase formulations of the present disclosure are co-administered with one or more other chemotherapeutic agents as part of a multi-agent chemotherapy regimen. In some embodiments, treating a patient with the L-asparaginase formulations of the present disclosure in addition to other agents helps ensure the availability of asparaginase to patients who have developed hypersensitivity to E. coli-derived asparaginase.

[0081] Examples of agents that may be part of a multi-agent chemotherapy regimen with the L-asparaginase of the present disclosure include, but are not limited to, cytarabine, vincristine, daunorubicin, methotrexate, leucovorin, doxorubicin, anthracyclines, corticosteroids and glucocortiods (including but not limited to prednisone, prednisolone, and / or dexamethasone), cyclophosphamide, 6-mercaptopurine, venetoclax, and etoposide. In some embodiments, the multi-agent chemotherapy regimen is L-asparaginase and one additional chemotherapeutic agent. In some embodiments, the multi-agent chemotherapy regimen is L-asparaginase and two or more additional chemotherapeutic agents.

[0082] As an example, an ALL patient would be co-administered the L-asparaginase of the present disclosure with a multi-agent chemotherapy during the three chemotherapy phases, including induction, consolidation, or intensification, and maintenance. In a specific example, the L-asparaginase formulation of the present disclosure is co-administered with an asparagine synthetase inhibitor (e.g., as described in WO 2007 / 103290, the entirety of which is incorporated herein by reference). In another specific example, the L-asparaginase formulation of the present disclosure is not co-administered with an asparagine synthetase inhibitor, but is co-administered with other chemotherapy agents. In another specific example, the L-asparaginase formulation of the present disclosure is co-administered with an asparagine synthetase inhibitor and other chemotherapy agents. The L-asparaginase formulation of the present disclosure may be co-administered before, after, or at the same time as other compounds as part of a multi-agent chemotherapy regimen. In a specific embodiment, the L-asparaginase of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens, more specifically an L-asparaginase comprising the sequence of SEQ ID NO:1.

[0083] The formulations described herein can be administered to patients as pharmaceutical compositions using standard techniques, which techniques and formulations can generally be found in Remington's Pharmaceutical Sciences, 22nd edition, Mack Publishing, 2015, incorporated herein by reference.

[0084] Suitable dosage forms depend, in part, on the method of use or route of entry, e.g., oral, transdermal, transmucosal, or by injection (parenteral). Such dosage forms should allow the therapeutic agent to reach target cells or otherwise have the desired therapeutic effect. For example, pharmaceutical compositions injected into the bloodstream are preferably soluble. The pharmaceutical compositions of the present disclosure may be formulated as pharmaceutically acceptable salts and complexes thereof. Pharmaceutically acceptable salts are non-toxic salts that exist in the amounts and concentrations at which they are administered. The preparation of such salts may alter the physical properties of the compound to facilitate pharmaceutical use without preventing it from exerting its physiological effect. Useful changes in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administration of higher concentrations of the drug. As will be appreciated by those of skill in the art, pharmaceutically acceptable salts of the modified proteins described herein may exist as complexes. 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 derived 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. When an acidic functional group is present, such as a carboxylic acid or a phenol, pharma- ceutically acceptable salts also include base addition salts, such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamines, and zinc. See, for example, Remington's Pharmaceutical Sciences (supra). Such salts can be prepared using the appropriate corresponding base.To facilitate administration of a particular asparaginase, pharma- ceutically acceptable carriers and / or excipients can also be incorporated into the pharmaceutical compositions of the present invention. Examples of carriers suitable for use in carrying out the present invention include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include sterile solutions of water for injection (WFI), saline, and dextrose.

[0085] The pharmaceutical composition of the present invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration. For oral administration, for example, the compound can be formulated into conventional oral dosage forms, such as capsules, tablets, and liquid preparations, such as syrups, elixirs, and concentrated drops. Alternatively, injections (parenteral administration), such as intramuscular, intravenous, intraperitoneal, and subcutaneous injections, may be used. For injections, the pharmaceutical composition is formulated in a solution, preferably in a physiologically compatible buffer or solution, such as, for example, saline, Hank's solution, or Ringer's solution. In certain embodiments, the L-asparaginase formulation is administered intramuscularly. In certain embodiments, the L-asparaginase formulation is administered intravenously. In certain embodiments, the L-asparaginase formulation is administered subcutaneously.

[0086] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are well known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, surfactants may be used to enhance penetration. Transmucosal administration may be via, for example, nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories. For topical administration, the compound can be formulated into ointments, salves, gels, or creams, as is well known in the art.

[0087] dosage In some embodiments, a dose is an amount administered to a human subject for a particular time and frequency. In some embodiments, a human subject with hypersensitivity is given a dose of an L-asparaginase formulation only if the hypersensitivity is attenuated.

[0088] In one exemplary embodiment, the L-asparaginase formulation is about 10 mg / m 2 ~100mg / m 2 In one exemplary embodiment, the L-asparaginase formulation is administered intramuscularly every other day for five consecutive days, followed by a rest period of two consecutive days, in an amount of about 25 mg / m 2 In one exemplary embodiment, the L-asparaginase formulation is administered intravenously every other day for five consecutive days, followed by a rest period of two consecutive days, in an amount of about 37.5 mg / m 2 In one exemplary embodiment, the L-asparaginase formulation is administered intravenously every other day for five consecutive days, followed by a rest period of two consecutive days, in an amount of about 50 mg / m 2 It is.

[0089] In further exemplary embodiments, and in accordance with any of the discussions herein regarding dosing, the L-asparaginase formulations administered at such doses are not conjugated to a polymer, such as a PEG moiety, and / or are not conjugated to a peptide containing only alanine and / or proline residues.

[0090] 1. Dose amount The amount of the L-asparaginase formulation of the present disclosure to be delivered will vary depending on many factors, such as the IC50, EC50, biological half-life of the compound, the age, size, weight, and health of the patient, and the disease or disorder being treated. The importance of these and other factors to be considered is well known to those of skill in the art. In general, for treating diseases including, but not limited to, ALL or LBL, the amount of the L-asparaginase formulation of the present disclosure is about 1 milligram per square meter (mg / m2) of the patient's body surface area. 2 ) to 1,000 / m2 The dose range is about 10 mg / m 2 ~about 100mg / m 2 Of course, other dosages and / or treatment regimens may be used, as determined by the attending physician.

[0091] In some embodiments, the method comprises administering a recombinant L-asparaginase of the present disclosure at a concentration of about 10 mg / m 2 ~about 100mg / m 2 In some embodiments, the method comprises administering an L-asparaginase formulation of the present disclosure in an amount of 10 mg / m 2 ~100mg / m 2 In some embodiments, the L-asparaginase formulations of the disclosure are 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 the equivalent thereof (e.g., on a protein content basis). In more specific embodiments, the L-asparaginase formulations of the present disclosure are administered at about 10, 20, 30, 40, 50, 60, 70, 80, 90, and about 100 mg / m 2 In another specific embodiment, the L-asparaginase formulation of the disclosure is administered in an amount selected from the group consisting of 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 In another specific embodiment, the L-asparaginase formulation of the disclosure is administered at a dose of about 300, 200 100, 95, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or 1 mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered at a dose of about 12 mg / m 2 ~about 90mg / m 2 In another exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 20 mg / m 2 ~about 80mg / m 2In another exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 37.5 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 37.5 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 37.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 100mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25 mg / m 2 ~about 80mg / m 2 is administered in an amount of

[0092] In some embodiments, the method comprises administering a recombinant L-asparaginase of the present disclosure at a concentration of about 25 mg / m 2 ~about 50mg / m 2 In some embodiments, the method comprises administering an L-asparaginase formulation of the present disclosure in an amount of 25 mg / m 2 ~50mg / m 2 In some embodiments, the L-asparaginase formulations of the disclosure are 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 2or its equivalent (eg, on a protein content basis).

[0093] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 20 mg / m 2 ~about 30mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 22.5 mg / m 2 ~about 28.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 23 mg / m 2 ~about 27mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 24 mg / m 2 ~about 26mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 24.5 mg / m 2 ~about 25.5mg / m 2 is administered in an amount of

[0094] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 40 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 42.5 mg / m 2 ~about 58.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 43 mg / m 2 ~about 57mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 44 mg / m 2 ~about 56mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 46 mg / m 2 ~about 54mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 47.5 mg / m 2 ~about 52.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 48 mg / m 2 ~about 52mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 49 mg / m 2 ~about 51mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 49.5 mg / m 2 ~about 50.5mg / m 2 is administered in an amount of

[0095] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 30 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 55mg / m 2 is administered in an amount of

[0096] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 40 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 70mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the recombinant L-asparaginase formulation of the present disclosure is administered in an amount of about 55 mg / m 2 ~about 60mg / m 2 is administered in an amount of

[0097] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 40 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 47.5 mg / m 2 ~about 50mg / m 2 is administered in an amount of

[0098] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 30 mg / m 2 ~about 35mg / m 2 is administered in an amount of

[0099] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 30 mg / m 2 ~about 95mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 90mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 85mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 75mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 55 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 60 mg / m 2 ~about 65mg / m 2 is administered in an amount of

[0100] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 30 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 50mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 42.5 mg / m 2 ~about 57.5mg / m 2 is administered in an amount of

[0101] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 30 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 35 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 40 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 45 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount of about 50 mg / m 2 ~about 55mg / m 2 is administered in an amount of

[0102] In some embodiments, the L-asparaginase formulation of the disclosure has a concentration of about 10 mg / m 2 ~about 50mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 12.5 mg / m 2 ~about 47.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 15 mg / m 2 ~about 45mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 20 mg / m 2 ~about 42.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 22.5 mg / m 2 ~about 40mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 24 mg / m 2 ~about 39mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 27 mg / m 2 ~about 37.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 30 mg / m 2 ~about 45mg / 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 In some embodiments, the recombinant L-asparaginase of the present disclosure is administered intramuscularly at a dose of 25 mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 25 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 37.5 mg / m 2 ~about 80mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 37.5 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 25 mg / m 2 ~about 37.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 30 mg / m 2 ~about 75mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 35 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 40 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 45 mg / m 2 ~about 60mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 50 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 40 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 45 mg / m 2 ~about 70mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 50 mg / m 2 ~about 65mg / m 2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 55 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 40 mg / m 2 ~about 60mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 45 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 47.5 mg / m 2 ~about 50mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 30 mg / m 2 ~about 35mg / m 2 It is administered intramuscularly in the amount of

[0103] In some embodiments, the L-asparaginase formulation of the disclosure has a concentration of about 10 mg / m 2 ~about 95mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 20 mg / m 2 ~about 60mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 22.5 mg / m 2 ~about 57.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 25 mg / m 2 ~about 55mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 27.5 mg / m 2 ~about 47.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 30 mg / m 2 ~about 45mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 32.5 mg / m 2 ~about 42.5mg / m 2 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 21.5 mg / m 2 ~about 38.5mg / m 2In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 36 mg / m 2 ~about 45mg / 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 In some embodiments, 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 It is administered intravenously in the amount of

[0104] In one exemplary embodiment, the L-asparaginase formulation of the present disclosure comprises about 25 mg / m 2 ~about 37.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 25 mg / m 2 ~about 100mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 25 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 25 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 30 mg / m 2 ~about 35mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 30 mg / m 2 ~about 95mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 35 mg / m 2 ~about 90mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 40 mg / m 2 ~about 85mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 45 mg / m 2 ~about 80mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 50 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 55 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 60 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 30 mg / m 2 ~about 60mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 35 mg / m 2 ~about 55mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 40 mg / m 2 ~about 50mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 42.5 mg / m 2 ~about 57.5mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 30 mg / m 2 ~about 75mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 35 mg / m 2 ~about 70mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 40 mg / m 2 ~about 65mg / m 2 In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 45 mg / m 2 ~about 60mg / m 2In one exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 50 mg / m 2 ~about 55mg / m 2 It is administered intravenously in the amount of

[0105] In another embodiment, the method includes administering a recombinant L-asparaginase of the present disclosure that elicits a reduced immunogenic response in a patient compared to non-recombinant L-asparaginase.

[0106] 2.Frequency of administration In certain embodiments, the treatment is at about 1 mg / m 2 ~about 1000mg / m 2 , typically about 10 mg / m 2 ~about 100mg / m 2 on a schedule ranging from about 3 times per week to about once per month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapeutic agents, including but not limited to glucocorticoids, corticosteroids, anti-cancer compounds, or other agents, such as, but not limited to, methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracyclines.

[0107] The L-asparaginase formulations of the present disclosure may be administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen. In certain embodiments, the L-asparaginase formulations of the present disclosure comprise a protein recombinantly produced in Pseudomonas fluorescens, and more specifically, the L-asparaginase formulation comprises the sequence of SEQ ID NO: 1. In some embodiments, the L-asparaginase formulations of the present disclosure are administered in a single dose for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) at a dose that depletes L-asparagine to undetectable levels using methods and equipment known in the art.

[0108] In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times per week. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week. In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times per week for at least one to three weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two 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 per week for about two weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day for five consecutive days for about two weeks, followed by a rest period of two consecutive days. 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 disclosure is administered three times per week for two weeks. In some embodiments, the L-asparaginase formulation of the disclosure is administered every other day for five consecutive days for two weeks, followed by a rest period of two consecutive days. In some embodiments, the L-asparaginase formulation of the 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 is free of a disease treatable by asparagine depletion. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days, continuing until the patient is free of a disease treatable by asparagine depletion. In some embodiments, the recombinant L-asparaginase of the present disclosure is administered on Monday, Wednesday, and Friday of the same week, continuing until the patient is free of a disease treatable by asparagine depletion.

[0112] In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week, continuing until the patient decides to terminate or postpone treatment. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day for five consecutive days, followed by a rest period of two consecutive days, continuing until the patient decides to terminate or postpone treatment. In some embodiments, 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 terminate 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 40-58 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 42-56 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 44-52 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 46-50 hours. In some embodiments, 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 64-80 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 66-78 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 68-76 hours. In some embodiments, the L-asparaginase formulations of the disclosure are administered about every 70 to 74 hours.

[0114] In some embodiments, the L-asparaginase formulations of the present disclosure are administered as a second-line treatment to patients who are hypersensitive to E. coli-derived L-asparaginase and / or may have had previous hypersensitivity to Erwinia chrysanthemi-derived L-asparaginase.

[0115] In some embodiments, the L-asparaginase formulation is administered to the human subject as a replacement for a dose of long-acting E. coli derived asparaginase. In some embodiments, six doses of the L-asparaginase formulation are administered to the human subject as a replacement for one dose of long-acting E. coli derived asparaginase. In some embodiments, seven doses of the L-asparaginase formulation are administered to the human subject as a replacement for one dose of long-acting E. coli derived asparaginase. In some embodiments, the long-acting E. coli derived asparaginase is pegaspargase. In further embodiments, six separate doses can occur over about two weeks. In yet another embodiment, seven separate doses can occur over about two weeks.

[0116] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, the cycle comprising a first dose, a second dose, and a third dose, which cycle is optionally repeatable, and wherein the first dose, the second dose, and the third dose are administered about 48-72 hours apart.

[0117] In some embodiments, the amount of the dose may vary within a cycle.

[0118] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 50 mg / m 2 This is followed by a rest period of two consecutive days.

[0119] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 37.5 mg / m 2 This is followed by a rest period of two consecutive days.

[0120] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle was 37.5 mg / m 2and the third dose of the cycle was 37.5 mg / m 2 This is followed by a rest period of two consecutive days.

[0121] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 37.5 mg / m 2 This is followed by a rest period of two consecutive days.

[0122] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 37.5 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 25 mg / m 2 followed by a rest period of 2 consecutive days. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0123] In some embodiments, the dose regimen of the L-asparaginase formulation comprises a cycle, which is optionally repeatable, and which comprises administration of the L-asparaginase formulation every other day for five consecutive days, followed by a rest period of two consecutive days, and the first dose of the cycle is 25 mg / m 2 and the second dose of the cycle is 25 mg / m 2 and the third dose of the cycle was 50 mg / m 2followed by a rest period of 2 consecutive days. In some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is administered on a Wednesday, and the third dose of the cycle is administered on a Friday.

[0124] In some embodiments, the disclosure provides a method for depleting asparaginase in a human subject to treat acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL), the method comprising administering about 25 mg / m L-asparaginase to a human subject on Monday, Wednesday, and Friday such that the human subject receives a total of 7 doses of L-asparaginase over a 2-week period. 2 of L-asparaginase administered intramuscularly.

[0125] In some embodiments, the 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) administering about 25 mg / m L-asparaginase to the human subject on Mondays and Wednesdays, such that the human subject receives a total of six doses of L-asparaginase over a two-week period; 2 of L-asparaginase administered intramuscularly to a human subject on Friday; and (b) administering about 50 mg / m 2 and administering L-asparaginase intramuscularly.

[0126] The dosing regimen may include any number of cycles over any number of weeks or until any endpoint specified herein.

[0127] In some embodiments, the L-asparaginase formulation of the disclosure is administered at 25 mg / m on (i) Mondays. 2 , (ii) 25 mg / m on Wednesday 2 , and (iii) 50 mg / m on Friday. 2 It is administered intravenously in the amount of

[0128] In some embodiments, the L-asparaginase formulation of the disclosure is at a dose of 25 mg / m every 48 hours. 2In some embodiments, the L-asparaginase formulation of the disclosure is administered intravenously in an amount of 37.5 mg / m every 48 hours. 2 In some embodiments, the L-asparaginase formulation of the disclosure is administered intravenously in an amount of 50 mg / m every 48 hours. 2 It is administered intravenously in the amount of

[0129] In some embodiments, the L-asparaginase formulation of the disclosure is at a dose of 25 mg / m every 48 hours. 2 In some embodiments, the L-asparaginase formulation of the disclosure is administered intramuscularly in an amount of 37.5 mg / m every 48 hours. 2 In some embodiments, the L-asparaginase formulation of the disclosure is administered intramuscularly in an amount of 50 mg / m every 48 hours. 2 It is administered intramuscularly in the amount of

[0130] In some embodiments, in conjunction with the above or below embodiments, the L-asparaginase formulation of the disclosure is administered at 25 mg / m on (i) Mondays 2 (ii) 25 mg / m on Wednesdays 2 (iii) 50 mg / m on Friday 2 It is administered intramuscularly in the amount of

[0131] For example, a dosing regimen can include 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 an L-asparaginase formulation over a two-week period, where during the first week, (i) on Monday, the first dose is 25 mg / m 2 or 37.5 mg / m 2 (ii) on Wednesday, a second dose of 25 mg / m 2 37.5 mg / m 2 (iii) on Friday, a third dose of 25 mg / m 2 37.5 mg / m 2 during the second week, (iv) on Monday, a fourth dose of 25 mg / m 2(v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2 It is administered intramuscularly in the amount of

[0132] In some embodiments, the dosing regimen comprises administration of six doses of the L-asparaginase formulation over a two week period, where during the first week, (i) on Monday, the first dose is 25 mg / m 2 (ii) on Wednesday, a second dose of 25 mg / m 2 (iii) on Friday, a third dose of 25 mg / m 2 during the second week, (iv) on Monday, a fourth dose of 25 mg / m 2 (v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2 It is administered intramuscularly in the amount of

[0133] In some embodiments, the dosing regimen comprises administration of six doses of the L-asparaginase formulation over a two week period, where during the first week, (i) on Monday, the first dose is 25 mg / m 2 (ii) on Wednesday, a second dose of 25 mg / m 2 (iii) on Friday, a third dose of 25 mg / m 2 during the second week; (iv) on Monday, a fourth dose of 25 mg / m 2 (v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2 It is administered intramuscularly in the amount of

[0134] In some embodiments, the dosing regimen comprises administration of six doses of the L-asparaginase formulation over a two week period, where during the first week, (i) on Monday, the first dose is 37.5 mg / m 2(ii) on Wednesday, a second dose of 37.5 mg / m 2 (iii) on Friday, a third dose of 37.5 mg / m 2 during the second week, (iv) on Monday, a fourth dose of 25 mg / m 2 (v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2 It is administered intramuscularly in the amount of

[0135] In some embodiments, the dosing regimen comprises administration of six doses of the L-asparaginase formulation over a two week period, where during the first week, (i) on Monday, the first dose is 37.5 mg / m 2 (ii) on Wednesday, a second dose of 37.5 mg / m 2 (iii) on Friday, a third dose of 37.5 mg / m 2 during the second week; (iv) on Monday, a fourth dose of 25 mg / m 2 (v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2 It is administered intramuscularly in the amount of

[0136] In some embodiments, the dosing regimen comprises administration of six doses of the L-asparaginase formulation over a two week period, where during the first week, (i) on Monday, the first dose is 25 mg / m 2 (ii) on Wednesday, a second dose of 25 mg / m 2 and (iii) on Friday, a third dose of 50 mg / m 2 during the second week, (iv) on Monday, a fourth dose of 25 mg / m 2 (v) on Wednesday, a fifth dose of 25 mg / m 2 (vi) on Friday, a sixth dose of 50 mg / m 2It is administered intramuscularly in the amount of

[0137] kit In one aspect, provided herein is a kit comprising (i) any one of the formulations described herein; and (ii) instructions for treating a disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof.

[0138] In some embodiments, the kit includes a formulation that includes (i) an L-asparaginase that includes four monomer units, each monomer unit having 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. In some embodiments, the L-asparaginase formulation includes less than about 0.6% low molecular weight (LMW) species after storage at 40° C. for 2 months. In some embodiments, the L-asparaginase formulation includes less than 2% high molecular weight (HMW) species after storage at 40° C. for 2 months. In some embodiments, the L-asparaginase formulation includes 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 2 months. In some embodiments, the L-asparaginase formulation includes 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 1 week.

[0139] In some embodiments, the kit includes a formulation comprising: (i) L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides comprising trehalose, sucrose, or any combination thereof; and (iii) one or more buffers substantially free of amino acids, wherein the formulation contains less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week.

[0140] In some embodiments, the kit includes a formulation comprising: (i) an L-asparaginase, the L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence at least about 70% identical to SEQ ID NO:1, the L-asparaginase being present at a concentration of about 20 mg / mL; (ii) trehalose present at a concentration of about 170 mM; (iii) sodium phosphate present at a concentration of about 20 mM; (iv) sodium chloride present at a concentration of about 50 mM; and (v) polysorbate 80 present at a concentration of about 0.02% (w / v), the formulation comprising less than about 0.6% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0141] In some embodiments, the kit includes a formulation comprising: (i) an L-asparaginase, the L-asparaginase comprising four monomer units, each monomer unit having an amino acid sequence at least about 95% identical to SEQ ID NO:1, the L-asparaginase being present at a concentration of about 20 mg / mL; (ii) trehalose present at a concentration of about 170 mM; (iii) sodium phosphate present at a concentration of about 20 mM; (iv) sodium chloride present at a concentration of about 50 mM; and (v) polysorbate 80 present at a concentration of about 0.02% (w / v), the formulation comprising less than about 5% low molecular weight (LMW) species after storage at 37° C. for 1 week. In some embodiments, the formulation has a pH of about 4.0 to about 8.5. In some embodiments, the formulation has a pH of about 7.0.

[0142] In some embodiments of the above, the kit comprises instructions for treating cancer. In some embodiments, the kit comprises instructions for treating acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is relapsed. In some embodiments, the kit comprises instructions for treating lymphoblastic lymphoma (LBL). In some embodiments, the LBL is relapsed. EXAMPLES

[0143] Nessler activity Asparaginase activity is measured by Nessler assay, where the measurement of asparaginase activity is based on an endpoint limit assay, where the enzyme is incubated at saturating L-asparagine concentrations for 15 min at +37° C. The reaction is stopped by adding Nessler's reagent, and the amount of ammonia produced during the reaction is evaluated colorimetrically (at 450 nm) using a calibration curve constructed from known amounts of ammonium sulfate.

[0144] Size Exclusion Chromatography (SEC) One hour prior to injection, the column was conditioned with 1 mL / min of mobile phase (50 mM phosphate, 200 mM NaCl, pH 7.0). BioRad Gel Filtration Standards were diluted 10-fold with Blue Dextran to a total volume of 1 mL. Samples and reference standards were diluted to 1 mg / mL in mobile phase and injected after multiple injections of a mobile phase blank to clean the column.

[0145] Please note that in the various tables of the Examples below, the entry "ND" indicates that the particular data point was not determined.

[0146] Example 1: Baseline buffer / excipient screening The effect of pH and buffer type from six buffer / pH combinations ranging from 4.5 to 8.0 on the stability of L-asparaginase was evaluated. An additional set of formulations with six different excipients in either 20 mM histidine buffer at pH 7.0 or 20 mM sodium phosphate at pH 8.0 was prepared as an initial excipient screen (Table 1). L-asparaginase (1.0 mL of the bulk pool of lots RM-LAP-P03 / P05 / P06 at 20.9 mg / mL) was buffer exchanged with 100 mL of the corresponding formulation buffer using a centrifugal ultrafiltration device (Amicon Ultra-4 10k MWCO). After the final buffer exchange, the protein concentration was measured and the samples were adjusted to approximately 20 mg / mL. The formulated samples were split into two aliquots and one-week stability studies were performed at 37°C and 5°C with the formulated RC. At the end of one week of storage, the formulations were analyzed simultaneously.

[0147] (Table 1) TIFF2024515555000004.tif175158

[0148] The objective of the excipient screening was to evaluate acetate, succinate, citrate, histidine, phosphate, and Tris buffers at pH 4.5-8.5. Additionally, the 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 excipient impact. SEC and Nessler activity assays were used to evaluate the stability of RC in the presence of these excipients in the selected buffers.

[0149] The purity of 26 formulations was assessed by SEC-HPLC. The results are summarized in Tables 2 and 3. For all formulations, little difference in SEC profile was observed between 5°C and 37°C conditions after 1 week of storage. However, differences were observed between different formulations. The main peak purity ranged from 66.4% to 98.3%. All histidine buffer formulations had purity levels between 66.4% and 74.0%, while the remaining formulations had purity levels between 90.6% and 98.3%. In histidine, succinate, citrate, lysine-containing phosphate, and arginine-containing phosphate buffers, LMW species were observed at about 11.6 min in the range of 1.3 to 32.4%. Formulations in acetate, phosphate, and Tris buffers showed LMW species between 0.2 and 0.6%. Formulations in phosphate buffer containing sodium chloride, trehalose, sorbitol, and sucrose showed levels of LMW species similar to the excipient-free formulation.

[0150] (Table 2) TIFF2024515555000005.tif166155

[0151] (Table 3) TIFF2024515555000006.tif126154

[0152] Based on the highest purity and lowest percentage of LMW species, acetate and phosphate buffers were selected for further evaluation. Additionally, trehalose, sorbitol, and sodium chloride were selected as excipients to be evaluated in the DOE to assess their impact on formulation stability under more severe stress conditions.

[0153] Example 2: DOE A statistical design of experiment (DOE) approach considering one numerical factor (pH) and one categorical factor (excipient) was used to evaluate the effect of acetate and phosphate buffer pH and excipients in the ranges of 4.5-5.5 and 7.0-8.0, respectively, on L-asparaginase. To accommodate the non-overlapping ranges of the two acetate and phosphate buffer systems, two independent DOEs using a linear one-factor design were performed simultaneously for each buffer system. The experimental design was generated using Design-Expert® version 9.0.6.2 from Stat Ease, Inc. The significance of the responses was evaluated at the 95% confidence level.

[0154] An additional set of off-design formulations evaluated the effect of three selected excipients using Tris buffer at pH 8.5, in DOEs with sorbitol and trehalose, and the effect of adding PS-80 in acetate at pH 5.3 and phosphate at pH 7.5. The formulations were subjected to heat stress at 40° C. for two weeks, and a single freeze / thaw cycle.

[0155] Nessler activity assays of formulations exposed to a single freeze / thaw cycle or stored at 40°C for 2 weeks had activities ranging from 600 to 1270 μmol / min.mgE (Tables 4 and 5). No significant loss of activity of the formulations was observed under thermal or freeze / thaw stress, and it is unclear whether formulations with increased activity are the result of the formulation buffer or the variability of the Nessler activity assay. Formulations were analyzed by RP-HPLC. By RP-HPLC, all formulations in both acetate and phosphate buffers were determined to be 100% major peak after a single freeze / thaw stress and 98% major peak and 2% minor peak after 2 weeks at 40°C.

[0156] (Table 4) TIFF2024515555000007.tif173156

[0157] (Table 5) TIFF2024515555000008.tif53155

[0158] All formulations and stress conditions were analyzed by SEC-HPLC. The results are shown in Table 6. After heat stress, the purity of the phosphate buffered formulations ranged from 88.70% to 99.31%, the HMW peaks ranged from 0.66% to 1.00%, no LMW shoulders were detected at 10.98%, and none of the LMW peaks were detected. At pH 7.0, all three excipients showed similar SEC profiles after heat stress, with the percentage of the main peak for each excipient being 99%.

[0159] (Table 6) TIFF2024515555000009.tif170155

[0160] The decrease in main peak purity and increase in LMW shoulder percentage were fitted to a linear model dependent on pH with only maximum main peak purity observed at pH 7.0 (Table 7). The off-design formulation at pH 8.5 also shows the formation of a large amount of LMW shoulder following heat stress (Table 8). The formation of HMW species was found to be excipient dependent rather than pH, with sorbitol having the greatest overall percentage of HMW species.

[0161] (Table 7) TIFF2024515555000010.tif28128

[0162] (Table 8) TIFF2024515555000011.tif90161

[0163] Under freeze / thaw stress for sodium phosphate formulations, the SEC-HPLC main peak purity ranged from 98.05% to 99.34%, the HMW peaks ranged from 0.66% to 0.71%, and the LMW peaks ranged from non-detectable to 1.28%. In trehalose formulations, no trends were found for SEC under freeze / thaw stress, and only the LMW peaks were observed.

[0164] Under freeze / thaw stress for sodium acetate formulations, the SEC-HPLC main peak purity ranged from 97.69% to 98.07%, the HMW peak ranged from 0.54% to 0.69%, and the LMW peak ranged from 1.34% to 1.62%. The results are shown in Table 9. No trends were found for SEC under freeze / thaw stress. Under heat stress, the purity for acetate formulations ranged from 97.65% to 98.02%, the HMW peak ranged from 0.46% to 0.68%, and the LMW peak ranged from 1.52% to 1.82%. Small differences in HMW percentages were found to be pH and excipient dependent, with HMW species being higher with sodium chloride than with trehalose or sorbitol at pH 5.5 and above.

[0165] (Table 9) TIFF2024515555000012.tif164152

[0166] All formulations and stress conditions were analyzed by IEX-HPLC. After heat stress, the phosphate buffer formulations showed a main peak purity range of 52.9%-63.1%, acidic species of 9.3%-18.8%, and basic species of 25.2%-30.1%. With increasing pH, the amount of acidic species increased while the main peak decreased. The percentage of the main peak also depended on the type of excipient. The results are shown in Tables 10 and 11.

[0167] (Table 10) TIFF2024515555000013.tif164152

[0168] (Table 11) TIFF2024515555000014.tif156157

[0169] At pH 7.0, the percentage of the main peak was 62.1-63.1%, 59.3-59.9%, and 57.2-57.5% for sodium chloride, trehalose, and sorbitol, respectively. No trends in the amount of basic species were observed under heat stress. Similar trends in pH were observed for the main peak and acidic species percentages for these formulations under freeze / thaw stress (Table 12), with little difference observed between excipients.

[0170] (Table 12) TIFF2024515555000015.tif155158

[0171] After heat stress, the purity of the acetate buffer formulations ranged from 51.3% to 56.4%, with 5.9% to 7.3% for acidic species and 37.2% to 41.8% for basic species (Table 11 above). The main peak decreased as the pH decreased, and the percentage of the main peak also depended on the type of excipient. At pH 5.0, the percentage of the main peak was 54.0%, 55.2%, and 52.1% for sorbitol, trehalose, and sodium chloride, respectively. No trends in the amount of basic species were observed under heat stress. Under freeze / thaw stress in sodium acetate buffer, similar trends were observed with pH and excipients as with heat stress, although the differences between formulations were relatively small (Table 12 above). The IEX-HPLC results of the off-design formulations are seen in Tables 13 and 14.

[0172] (Table 13) TIFF2024515555000016.tif49159

[0173] (Table 14) TIFF2024515555000017.tif88160

[0174] All formulations were colorless and clear after being placed in the vial. After freeze / thaw stress, all formulations were colorless and slightly milky (Tables 15, 16, and 17). Under heat stress for sodium phosphate formulations, product-associated precipitates were observed for sorbitol at pH 8.0, trehalose at pH 7.5 and 8.0, and all sodium chloride formulations. For sodium acetate formulations, particulates were observed for all formulations except 250 mM sorbitol at pH 4.5 and 250 mM trehalose at pH 5.5.

[0175] (Table 15) TIFF2024515555000018.tif156156

[0176] (Table 16) TIFF2024515555000019.tif151155

[0177] (Table 17) TIFF2024515555000020.tif89161

[0178] Analytical data for sodium phosphate buffered formulations under heat stress were selected for further analysis based on the degree of variation between formulations and the client's historical experience with RC in phosphate buffer. Results from SEC, IEX, and visual testing showed clear trends with pH on formulation stability, with excipients showing reduced effectiveness for the pH 7.0 formulation.

[0179] Numerical optimization was performed on sodium phosphate formulations under heat stress using Design-Expert® version 9.0.6.2 from Stat-Ease, Inc. to maximize the main peak percentage in SEC and IEX and minimize the formation of LMW and HMW species in SEC and acidic and basic species in IEX. Numerical optimization was performed on the analytical results from the 20 mM sodium phosphate DOE after 2 weeks of heat stress. Formulation optimization was performed to find the optimal formulation without constraints on pH or excipients. The optimization parameters and results are found in Tables 18, 19, 20, and 21. When the presence of particulates in the formulation was used as the response and all results were given equal importance, the optimal formulation was calculated to be 20 mM sodium phosphate, 250 mM trehalose at pH 7.2. When the appearance of particulates in the formulation was not a factor as a constraint in the optimization and all results were given equal importance, the optimal formulation was calculated to be 20 mM sodium phosphate, 150 mM sodium chloride at pH 7.0. Based on the DOE and non-DOE formulations, 20 mM sodium phosphate, 250 mM trehalose, pH 7.0 at 20 mg / mL RC was selected for further testing in surfactant studies.

[0180] (Table 18) TIFF2024515555000021.tif46146

[0181] (Table 19) TIFF2024515555000022.tif31164

[0182] (Table 20) TIFF2024515555000023.tif56164

[0183] (Table 21) TIFF2024515555000024.tif23164

[0184] Example 3: Surfactant Studies A surfactant screening study was performed to evaluate the effect of PS-80 concentration on the opalescence observed after freeze / thaw or agitation stress. L-asparaginase was formulated in 20 mM sodium phosphate at pH 6.5, 7.0, and 7.5 with 250 mM trehalose at PS-80 concentrations ranging from 0.00% to 0.06% w / v (Table 22). Alternative formulations include 20 mM sodium acetate, 250 mM trehalose at pH 5.5 with PS-80 at 0.00% and 0.04% w / v.

[0185] (Table 22) TIFF2024515555000025.tif195154TIFF2024515555000026.tif83154

[0186] L-Asparaginase (1760 mg of 22 mg / mL) was dialyzed into the corresponding formulation buffer without PS-80 using a 10K MWCO Slide-A-Lyzer G2 dialysis cassette. Dialysis was performed at room temperature with a minimum of 40:1 formulation buffer to sample volume. Four dialysis buffer exchanges were performed. After dialysis, the material was removed from the cassette and the concentration and pH were measured. The material was then divided and PS-80 was added to the appropriate concentration using a 10% (w / v) PS-80 solution in H2O. After mixing, each formulation was equally aliquoted into six 2 mL vials.

[0187] During freeze / thaw and agitation evaluation, one vial from each formulation was kept at 5°C for first time point testing (8 in total). One and three freeze / thaw cycles were performed independently on separate vials of each formulation. Each freeze / thaw cycle consisted of freezing at -75±10°C for a minimum of 2 hours and thawing at room temperature for a minimum of 2 hours and until completely thawed. Three vials from each formulation were required for agitation evaluation (24 in total). One vial was agitated on a rotator at 60 rpm for 24 hours at ambient temperature, one vial was agitated under the same conditions for 48 hours, and one vial was placed next to the rotator during the study as a control.

[0188] Surfactant studies were conducted to evaluate the effect of PS-80 on opalescence formation under freeze / thaw or agitation stress for RC formulations in 20 mM phosphate, 250 mM trehalose at pH 6.5, 7.0, and 7.5 with 0.00%, 0.02%, 0.04%, and 0.06% PS-80 (Table 22). As potential buffer alternatives, two additional formulations were added to the study: 20 mM acetate, 250 mM trehalose at pH 5.5 with 0.00% and 0.04% PS-80. The formulations were then subjected to one and three freeze / thaw cycles at -75°C and stressed with agitation at room temperature for 24 and 48 hours. Controls of each formulation were kept without agitation and at 5°C for freeze / thaw stress and at room temperature for 48 hours.

[0189] By visual examination, all control and stressed samples were colorless and slightly opalescent with no product associated particulates. No differences in the degree of opalescence were observed between the control and freeze / thaw and agitation samples or between the different levels of PS-80. The protein concentration of the control and stressed formulations was also evaluated. Protein concentrations ranged from 19 to 22 mg / mL (Table 22). No significant changes or trends were observed upon freeze / thaw or agitation stress.

[0190] DLS analysis showed that all samples had a hydrodynamic radius between 4.1 and 4.5, with a polydispersity percentage between 10.7% and 19.4% (Tables 23 and 24). No significant differences or trends in DLS were observed for formulation or stress condition.

[0191] (Table 23) TIFF2024515555000027.tif213109

[0192] (Table 24) TIFF2024515555000028.tif82128

[0193] SEC-HPLC results show that regardless of stress, all formulations in 20 mM phosphate buffer had similar profiles before and after stress conditions with a main peak percentage of 99.3% and HMW peaks of 0.7% (Table 25). Similarly, SEC-HPLC of the 20 mM acetate formulations showed 0.7% HMW peaks, 97.9%-98.1% main peaks, and 1.3%-1.4% LMW peaks (Table 26).

[0194] (Table 25) TIFF2024515555000029.tif201164

[0195] (Table 26) TIFF2024515555000030.tif78149

[0196] Sub-visible particulates were measured by HIAC for TO, 1 freeze / thaw cycle, 3 freeze / thaw cycle, agitated control, 24 hour agitated, and 48 hour agitated samples with various PS-80 concentrations. The results are summarized in Tables 27 and 28. For all samples, the cumulative coefficients / mL ranged from 2330 to 45270, 390 to 35857, 78 to 6430, and 0 to 150 for ≧2 μm, 5 μm, ≧10 μm, and ≧25 μm, respectively. Although some conditions had increased levels of particulates compared to other conditions in the same formulation, these samples are considered outliers rather than representing a trend. For example, the 20 mM phosphate, 250 mM trehalose, 0.04% PS-80, pH 7.0 solution had 2842 ≧10 μm particles at the initial time point, whereas only 155 particles were present in the agitated control.

[0197] (Table 27) TIFF2024515555000031.tif189160 1 Testing of the sample was interrupted and not fully completed, however the measurement(s) taken indicate the actual result of the sample.

[0198] (Table 28) TIFF2024515555000032.tif107154

[0199] IEX-HPLC was performed on all 20 mM phosphate formulations at all conditions. The main peak percentages for the TO control at pH 7.0 with 0%, 0.02%, 0.04%, and 0.06% PS-80 were 68.88%, 69.83%, 71.36%, and 70.38%, respectively (Table 29). At 0.04% PS-80, the main peak percentages for the TO controls at pH 6.5, 7.0, and 7.5 were 70.11%, 71.36%, and 68.04%, respectively. No significant changes were observed in the IEX profiles of all formulations after exposure to freeze / thaw or agitation stress. These results indicate that there is little benefit to the addition of PS-80 below 0.06% w / v.

[0200] (Table 29) TIFF2024515555000033.tif199157

[0201] Example 4: Salt / Trehalose Dependence Studies A preformed salt / trehalose dependency study performed with L-asparaginase at 22 mg / mL was formulated in nine different formulation buffers according to Table 30. An aliquot of 27 mL lot RE-LAP-P59 was split into six portions of approximately 3 mL (3 portions) and 6 mL (3 portions). Buffer exchange by dialysis was performed for formulations 1-6 using a 10K MWCO Slide-A-Lyzer G2 cassette. Dialysis was performed at room temperature with 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 being overnight.

[0202] (Table 30) TIFF2024515555000034.tif50163

[0203] Following dialysis, the material was removed from the dialysis cassette and pH and protein concentration were verified. 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 divided into equal aliquots of 3 mL 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 placed in vials in a safety cabinet. One set of formulations was stored at 5°C until testing for initial material quality evaluation, one set was stored at 5°C for 3 weeks, and one set was subjected to heat stress at 40°C for 3 weeks. The 3-week time point was selected after visual evaluation after 1.5 weeks of storage at 40°C. Formulations 1 and 2 at 5°C were further tested by IEX and SEC HPLC after 7 weeks.

[0204] A salt / trehalose dependency study was performed to evaluate the effect 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). Formulations were exposed to 5° C. and 40° C. for 1.5 and 3 weeks. Size exclusion and IEX chromatography were used to evaluate the stability of RC in the presence of these various salt concentrations in selected buffers. Osmolality and conductivity were also determined at early time points and are shown in Table 31.

[0205] (Table 31) TIFF2024515555000035.tif58146

[0206] Upon visual examination, all initial and stressed samples were colorless and slightly milky with little to no product associated particulates. All formulations became more milky upon stress. No particles were present in the formulations containing 0.02% PS-80 regardless of storage time (Table 32). This result indicates that the addition of 0.02% PS-80 may retard the formation of particles during long term storage. There was no dependency of appearance on salt concentration. Protein concentration of initial and stressed formulations was also evaluated. Protein concentrations ranged from 19.7 to 25.2 mg / mL (Table 33). No significant changes or trends in the stressed samples were observed.

[0207] (Table 32) TIFF2024515555000036.tif21398

[0208] (Table 33) TIFF2024515555000037.tif154164

[0209] IEX-HPLC results at early time points showed similar profiles with the main peak percentages ranging from 64.1% to 66.4%, the acidic species percentages ranging from 5.9% to 7.5%, and the basic species percentages ranging from 27.7% to 28.7%. After 3 weeks at 40°C, samples showed significant differences in charge heterogeneity profiles with the main peak percentages ranging from 35.0% to 42.3%, the acidic species percentages ranging from 22.5% to 32.0%, and the basic species percentages ranging from 33.0% to 36.2%. After 7 weeks at 5°C, only minor variations were observed with the main peak percentages ranging from 59.3% to 61.3%, the acidic species percentages ranging from 9.7% to 12.8%, and the basic species percentages ranging from 27.9% to 29.0% (Table 34). The salt-containing formulation has a slightly higher main peak percentage compared to the salt-free formulation. The addition of 0.02% PS-80 provided little or no benefit in terms of charge heterogeneity.

[0210] (Table 34) TIFF2024515555000038.tif87164 1 Samples were not analyzed until seven weeks after the start of the study.

[0211] SEC-HPLC results at early time points showed similar profiles with main peak percentages ranging from 98.0% to 98.2% and HMW peak percentages ranging from 1.9% to 2.0%. After 3 weeks at 40°C, samples showed a slight degradation in purity due to the appearance of LMW peaks. The main peak percentages for samples stressed for 3 weeks were 94.8% to 95.4%, HMW peak percentages ranging from 1.9% to 2.1%, and LMW peak percentages ranging from 2.7% to 3.1%. After 3 weeks at 5°C, no significant changes in the main and HMW peak percentages were observed (Table 35). High and low molecular weight species were minimized by the addition of salt and no benefit was observed when the formulation was supplemented with 0.02% PS-80.

[0212] (Table 35) TIFF2024515555000039.tif87164

[0213] Example 5: Comparison of recombinant crisantaspase (RC-P) from Pseudomonas with Erwinase Experiments were conducted to compare the relevant quality attributes of recombinant crisantaspase from Pseudomonas (RC-P) and Erwinase. A summary of the data and results comparing RC-P with Erwinase to show equivalence is shown in Table 36. The results show that RC-P and Erwinase are comparable in terms of their structure and enzymatic activity. Differences in size and charge were observed.

[0214] Table 36. Summary of equivalence results TIFF2024515555000040.tif29160TIFF2024515555000041.tif249160

[0215] MS data mapping peptides were mined for common or known modifications containing peptides, including oxidation, deamidation, succinimide, isomerization, glycosylation, methylation, and acetylation. The relative extent of each modification was calculated using the counts of modified peptides normalized to the total modified and unmodified peptides. The relative levels reported herein were semi-quantitative. Methylation and acetylation modifications were not seen in either material. Oxidation, deamidation, and succinimide were seen in both RC-P and Erwinase. As shown in Table 37, these levels were comparable and relatively low (Note: PTMs less than 0.5% are not reported). Furthermore, low levels of glycosylation were observed at multiple lysine positions only in Erwinase. The additional glycosylation modifications in Erwinase affect its charge properties, resulting in an increase in its acidic variants. Overall, both RC-P and Erwinase have established primary amino acid structures. These PTMs are comparable except for the additional glycosylation modifications observed in Erwinase.

[0216] Table 37. Observed post-translational modifications TIFF2024515555000042.tif75160

[0217] Size exclusion chromatography (SE-UHPLC) provides quantitative information on the molecular size distribution of native proteins. As shown in Table 38, the SE-UHPLC profiles of RC-P and Erwinase showed the relative peak area distribution of the size exclusion peaks. Both RC-P and Erwinase had a predominant main peak content. The amount of high molecular weight species (HMW) observed in Erwinase (7.3%) was higher than that of RC-P (0.2%). The amount of low molecular weight species (LMW) observed in both RC-P (0.1%) and Erwinase (0.3%) was comparable.

[0218] Table 38. HMW, main peak, and LMW percent from SE-UHPLC analysis TIFF2024515555000043.tif29160

[0219] Size exclusion chromatography with multi-angle laser light scattering: SE-HPLC_MALLS was performed for both RC-P and Erwinase. The size exclusion peak identification is listed in Table 39. SE-HPLC separation coupled with MALLS performed for both RC-P and Erwinase identified the major content of SEC as tetramer with molecular weight in the range of 133-134 kDa. Both materials showed predominant tetramer content. The HMW1 species of both materials was identified as an octamer with MW in the range of 277-286 kDa, which was close to the theoretical octamer mass of 280 kDa. The HMW2 species observed only for Erwinase was identified as a hexadecamer (16-mer) with MW of 554 kDa, which was consistent with the theoretical hexadecamer mass of 560 kDa. The size of the LMW species could not be accurately assessed by MALLS for both RC-P and Erwinase due to the very low abundance of these forms. SEC-MALLS results showed that both RC-P and Erwinase contained predominantly tetrameric forms, and their HMW forms were similar and identified as octamers. Furthermore, Erwinase contained low levels of 16-mers.

[0220] Table 39: SEC-MALLS results TIFF2024515555000044.tif66160

[0221] Imaging Capillary Isoelectric Focusing (iCIEF): iCIEF is an orthogonal assay that provides a quantitative measurement of the charge heterogeneity of RC-P proteins. The iCIEF profiles of RC-P and Erwinase, the relative peak areas of the main peak, acidic groups, and basic groups relative to the total area are listed in Table 40. Both materials showed very comparable pI values ​​in terms of main content. Erwinase had a higher acidic variant content.

[0222] Table 40. Distribution of iCIEF charge variants of RC-P and Erwinase TIFF2024515555000045.tif43160Note: Due to rounding, the total percentage may not add up to exactly 100%.

[0223] Oxidation Studies: Both RC-P and Erwinase samples were subjected to forced oxidation by treating with 0.01% (v / v) H2O2 for 4 h at room temperature, respectively. Analytical test results showed that the oxidized RC-P and Erwinase samples had a total oxidation of around 30% of methionine residues, an increase of about 20% in pre-peak content in RP-UHPLC, an increase in acidic variants in iCIEF, a slight increase in HMW content in SEC, and had comparable CIEX, HIC, and activity properties. Detailed analytical results are described below.

[0224] Peptide mapping results: Tryptic peptide mapping LC-MS method was used to confirm the primary structure of the protein and to identify potential post-translational modifications induced by forced oxidation conditions, especially methionine oxidation. Peptide mapping results showed that forced oxidized RC-P and Erwinase had the expected primary structure with methionine oxidation at multiple positions (Table 41). Overlays of peptide mapping profiles of RC-P and Erwinase treated with H2O2 are comparable. Traces of both oxidized samples showed increased peak intensities of oxidized peptides containing M133, M60, and M121, respectively. RC-P and Erwinase treated with H2O2 showed various levels of oxidation at multiple methionine positions (Table 41). The oxidation-prone methionine positions, from most to least oxidized, are M133 (~20%)>M60 (~8%)>M121 (~4%)>M308 (~1%). Total methionine oxidation of around 33% was observed in both samples. Similar levels of deamidation and succinimide intermediates at Asn281 were observed in both oxidized samples and the reference standard.

[0225] Table 41. PTMs of oxidized RC-P and Erwinase TIFF2024515555000046.tif98160

[0226] SE-UHPLC Results: The SE-UHPLC method was used to evaluate the size distribution of native RC-P under non-denaturing conditions. An overlay of oxidized RC-P, oxidized Erwinase, and the reference standard was analyzed. No significant profile differences were observed between the oxidized samples and RS. Both oxidized samples showed a predominantly tetramer peak, a slight increase in HMW content, and comparable LMW content compared to the reference standard. The relative peak intensities are listed in Table 42.

[0227] Table 42 SE-UHPLC results TIFF2024515555000047.tif57160

[0228] CIEX Results: The charge distribution of native RC-P under non-denaturing conditions was evaluated using the CIEX method. An overlay of oxidized RC-P, oxidized Erwinase, and the reference standard was analyzed. There was no significant profile change between the oxidized samples and the reference standard. Both the oxidized samples showed mainly main peak content. The relative peak intensities of the stressed samples and the reference standard are listed in Table 43.

[0229] Table 43: CIEX results TIFF2024515555000048.tif62160

[0230] iCIEF Results: The iCIEF method is an orthogonal method for evaluating the charge distribution of RC-P subjected to an electric field. Overlays of oxidized samples and reference standards were analyzed. Their relative peak intensities are listed in Table 44. Both oxidized samples showed an increase in the acidic peak with a pI around 8.5. Oxidation has no effect on the basic variant.

[0231] Table 44. iCIEF results from oxidation samples and RS TIFF2024515555000049.tif62160

[0232] Low pH research: Both RC-P and Erwinase samples were subjected to low pH treatment with 50 mM sodium phosphate at pH 3.4 at room temperature, followed by freezing at -80°C. Previous studies have shown that the incubation time at pH 3.4, from 15 min to 7 days, was not critical, and the SE-UHPLC profiles of the treated samples were comparable. The treated samples were relatively stable for storage at -80°C for up to 8 weeks. Analytical test results showed that the treated RC-P and Erwinase samples had a monomer content around 10% and reduced activity. Their peptide mapping profiles, PTM, CIEX, RP-UHPLC, and HIC characteristics were comparable to those of the untreated ones. Detailed analytical results are described below.

[0233] SE-UHPLC Results: The SE-UHPLC method is used to evaluate the size distribution of RC-P in its native state under non-denaturing conditions. An overlay of low pH treated RC-P, Erwinase, and the reference standard was analyzed. Both low pH treated RC-P and Erwinase showed a predominantly tetramer peak and an increase in LMW content of around 10% compared to the reference standard. Low pH treatment resulted in a slight increase in HMW content from 7.3% to 10.6% for Erwinase, but no effect on RC-P. These relative peak intensities are listed in Table 45.

[0234] Table 45. SE-UHPLC results TIFF2024515555000050.tif62160

[0235] CIEX Results: The CIEX method is used to evaluate the charge distribution of RC-P in the native state under non-denaturing conditions. An overlay of RC-P treated at low pH, Erwinase, and the reference standard was analyzed. There was no significant profile change between the pH treated samples and the reference standard. Both treated samples showed mainly main peak content. The relative peak intensities of the treated samples and the reference standard are listed in Table 46.

[0236] Table 46: CIEX results TIFF2024515555000051.tif62160

[0237] iCIEF Results: The iCIEF method is an orthogonal method to evaluate the charge distribution of RC-P subjected to an electric field. An overlay of RC-P treated at low pH, Erwinase, and a reference standard was analyzed. Their relative peak intensities are listed in Table 47. Both RC-P and Erwinase treated at low pH showed an increase in an acidic peak with a pI around 7.2, which was previously identified as a monomer. Low pH treatment had no effect on the basic variant.

[0238] Table 47. iCIEF results from oxidation samples and RS TIFF2024515555000052.tif62160

[0239] The equivalence of RC-P and Erwinase was assessed by comparing their physicochemical, purity, structural, and potency properties. Size-based heterogeneity analyzed by SE-UHPLC, SEC-MALLS, and SEC-MS was comparable. Both materials have a predominant tetrameric structure. The HMW species was identified as the octamer. Erwinase has a higher level of the octamer. Low levels of the 16-mer species were also observed in Erwinase (<2%). It has been previously demonstrated through size variant characterization of RC-P that the octamer exhibits comparable activity to the tetramer. Overall, the differences in size distribution observed between Erwinase and RC-P have minimal impact on drug potency. Erwinase has glycosylation modifications that contribute to the differences in acidic variants. The higher levels of octamer / 16-mer observed in Erwinase contributed to the late-eluting basic variants shown on CIEX. Previous forced degradation studies of RC-P demonstrate that glycated RC-P has comparable structure and activity to RC-P. The hydrophobicity profiles measured by HIC and RP-UHPLC are comparable for both materials. The slight differences observed are due to the glycosylation modification and higher level of HMW content of Erwinase. The primary and higher order structures of both materials are comparable. The potency results are comparable. Forced degradation studies show that Erwinase and RC-P have comparable stability under oxidative and low pH stress. Both oxidized RC-P and Erwinase, which have an increase in the RP-UHPLC pre-peak of around 20%, have comparable activity compared to these untreated materials. Both RC-P and Erwinase treated with low pH stress conditions have a reduction in activity, which correlates very well with the monomer content of the treated samples. Based on these data, RC-P exhibits comparable structure, biological properties, and function to Erwinase.

[0240] Enumerated Embodiments The embodiments listed below represent some aspects of the present invention.

[0241] Embodiment 1: An aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence that is at least about 95% identical to SEQ ID NO:1; (ii) one or more disaccharides, including trehalose, sucrose, or any combination thereof; (iii) one or more buffers that are substantially free of amino acids; and after storage at 37° C. for 1 week, contains less than about 5% low molecular weight (LMW) species.

[0242] Embodiment 2: The formulation of embodiment 1, wherein the L-asparaginase is present in a concentration of about 20 mg / mL.

[0243] Embodiment 3: The formulation of embodiment 1 or embodiment 2, wherein the L-asparaginase is non-PEGylated and non-PASylated.

[0244] Embodiment 4: The formulation of any one of embodiments 1-3, wherein the one or more disaccharides comprises trehalose.

[0245] Embodiment 5: The formulation of embodiment 4, wherein trehalose is present at a concentration of about 50 mM to about 300 mM.

[0246] Embodiment 6: The formulation of embodiment 4, wherein trehalose is present at a concentration of about 150 mM to about 275 mM.

[0247] Embodiment 7: The formulation of embodiment 4, wherein the trehalose is present at a concentration of about 170 mM.

[0248] 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.

[0249] Embodiment 9: The formulation of any one of embodiments 1 to 7, wherein the one or more buffers comprises a phosphate buffer.

[0250] Embodiment 10: The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise sodium phosphate.

[0251] Embodiment 11: The formulation of embodiment 10, wherein sodium phosphate is present at a concentration of about 0.5 mM to about 50 mM.

[0252] Embodiment 12: The formulation of embodiment 10, wherein the sodium phosphate is present at a concentration of about 20 mM.

[0253] Embodiment 13: The formulation of any one of embodiments 1 to 12, further comprising sodium chloride.

[0254] Embodiment 14: The formulation of embodiment 13, wherein sodium chloride is present at a concentration of about 25 mM to about 150 mM.

[0255] Embodiment 15: The formulation of embodiment 13, wherein sodium chloride is present at a concentration of about 50 mM.

[0256] Embodiment 16: The formulation of any one of embodiments 1 to 15, further comprising one or more excipients.

[0257] Embodiment 17: The formulation of embodiment 16, wherein the one or more excipients comprises polysorbate 80.

[0258] Embodiment 18: The formulation of embodiment 17, wherein polysorbate 80 is present in a concentration of about 0.004% (w / v) to about 0.2% (w / v).

[0259] Embodiment 19: The formulation of embodiment 17, wherein polysorbate 80 is present in a concentration of about 0.02% (w / v).

[0260] Embodiment 20: The formulation of any one of embodiments 1 to 19, having a pH of about 4.0 to about 8.5.

[0261] Embodiment 21: The formulation of any one of embodiments 1 to 19, having a pH of about 7.0.

[0262] Embodiment 22: A method of treating a disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject a formulation of any one of embodiments 1-21.

[0263] Embodiment 23: The method of embodiment 22, wherein the disease, condition, or disorder is cancer.

[0264] Embodiment 24: The method of embodiment 23, wherein the cancer is acute lymphoblastic leukemia (ALL).

[0265] Embodiment 25: The method of embodiment 24, wherein the ALL is relapsed ALL.

[0266] Embodiment 26: The method of embodiment 23, wherein the cancer is lymphoblastic lymphoma (LBL).

[0267] Embodiment 27: The method of embodiment 26, wherein the LBL is recurrent LBL.

[0268] Embodiment 28: The method of any one of embodiments 22-27, wherein the formulation is administered intramuscularly.

[0269] Embodiment 29: The method of any one of embodiments 22-27, wherein the formulation is administered intravenously.

[0270] Embodiment 30: The method of any one of embodiments 22-29, wherein the formulation is co-administered with one or more other chemotherapeutic agents.

[0271] Embodiment 31: (i) a formulation according to any one of embodiments 1 to 21; (ii) instructions for treating a disease, condition, or disorder treatable by asparagine depletion in a subject in need thereof. Including the kit.

[0272] Embodiment 32: The kit of embodiment 31, wherein the disease, condition, or disorder is cancer.

[0273] Embodiment 33: The kit of embodiment 32, wherein the cancer is acute lymphoblastic leukemia (ALL).

[0274] Embodiment 34: The kit of embodiment 33, wherein the ALL is relapsed ALL.

[0275] Embodiment 35: The kit of embodiment 32, wherein the cancer is lymphoblastic lymphoma (LBL).

[0276] Embodiment 36: The kit of embodiment 35, wherein the LBL is recurrent LBL.

Claims

1. 1. An aqueous, non-lyophilized formulation comprising: (i) an L-asparaginase comprising four monomeric units, each monomeric unit having an amino acid sequence that is at least about 70% identical to SEQ ID NO:1; (ii) a surfactant, and (iii) one or more stabilizers, or one or more buffers; The formulation comprising:

2. 10. The formulation of claim 1, wherein the one or more stabilizers comprise one or more disaccharides, one or more sorbitol, one or more amino acids, or any combination thereof.

3. 3. The formulation of claim 2, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof. (a) containing 0.6% low molecular weight (LMW) species; and / or (b) containing less than 2% high molecular weight (HMW) species; The formulation of claim 1. (a) the L-asparaginase is present at a concentration of 20 mg / mL; and / or (b) the L-asparaginase is non-PEGylated and non-PASylated; The formulation of claim 1. (a) the one or more buffers comprise a phosphate buffer, an acetate buffer, or any combination thereof; and / or (b) one or more buffers are substantially free of amino acids; The formulation of claim 1.

7. 7. The formulation of claim 6, wherein the one or more buffers comprise sodium phosphate. (a) the sodium phosphate is disodium phosphate (anhydrous), monosodium phosphate monohydrate, or a combination thereof; or (b) the sodium phosphate is present at a concentration of 0.5 mM to 50 mM; The formulation of claim 7.

9. 10. The formulation of claim 1 further comprising sodium chloride.

10. 10. The formulation of claim 9, wherein the sodium chloride is present at a concentration of 25 mM to 150 mM.

11. The formulation of claim 1 further comprising one or more excipients.

12. (a) the one or more excipients comprise polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof; or (b) the one or more excipients are present in a concentration of from 0.004% (w / v) to 0.3% (w / v); The formulation of claim 11.

13. The formulation of claim 1 having a pH of 4.0 to 8.

5.

14. A medicament comprising the formulation of any one of claims 1 to 13 for use in a method for treating a disease, condition, or disorder treatable by asparagine depletion in a subject, comprising: The pharmaceutical, wherein the method comprises administering the formulation to a subject.

15. The pharmaceutical of claim 14 , wherein the disease, condition, or disorder is cancer.

16. The pharmaceutical composition of claim 15, wherein the cancer is acute lymphoblastic leukemia (ALL).

17. The pharmaceutical composition of claim 16, wherein the ALL is recurrent ALL.

18. The pharmaceutical composition of claim 15, wherein the cancer is lymphoblastic lymphoma (LBL).

19. The pharmaceutical composition of claim 18, wherein the LBL is recurrent LBL.

20. The pharmaceutical described in claim 14, wherein in the method, the formulation is administered intramuscularly, intravenously or subcutaneously.

21. The pharmaceutical composition of claim 14, wherein the method comprises administering the formulation simultaneously with one or more other chemotherapeutic agents.

22. (i) a formulation according to any one of claims 1 to 13; (ii) instructions for treating a disease, condition, or disorder treatable by asparagine depletion. Including the kit.

23. (i) a formulation according to any one of claims 1 to 13; (ii) one or more pharma- ceutically acceptable excipients; 4. A unit dosage form comprising: