CA3213726A1

CA3213726A1 - Formulations of l-asparaginase

Info

Publication number: CA3213726A1
Application number: CA3213726A
Authority: CA
Inventors: Sekhar Kanapuram; Haripada MAITY; Guifeng Jiang; Roja NARWAL
Original assignee: Jazz Pharmaceuticals Ireland Ltd
Current assignee: Jazz Pharmaceuticals Ireland Ltd
Priority date: 2021-04-06
Filing date: 2022-04-05
Publication date: 2022-10-13
Also published as: JP2024515555A; CN117377494A; AU2022256071A1; MX2023011693A; CL2023002983A1; CO2023014093A2; BR112023019862A2; WO2022217231A1; AR125301A1; EP4319818A1; KR20230165799A

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 (for example, cancers such as Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LBL)) that are treatable by asparagine depletion.

Description

FORMULATIONS OF L-ASPARAGINASE
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63/171,429, filed April 6, 2021, the disclosure of which is hereby incorporated herein by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 210462000340SEQLIST.TXT, date recorded: April 5, 2022, size: 3,096 bytes).
BACKGROUND OF THE INVENTION

[0003] Proteins with L-asparagine aminohydrolase activity, commonly known as L-asparaginases, have successfully been used for the treatment of various diseases that are potentially fatal, including cancers such as Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LBL), for which children constitute a large proportion of patients stricken with these diseases.
100041 L-asparaginases of bacterial origin have a high immunogenic and antigenic potential. Currently on the market as first line treatment are E. coil derived L-asparaginase and pegaspargase. These products can provoke adverse hypersensitivity reactions including allergic reaction, silent inactivation, and anaphylactic shock in patients. Patients who experience hypersensitivity reactions to these products often have to stop treatment, resulting in poorer prognosis and survival rates. These patients have turned to ErwinazeilD after experiencing hypersensitivity reactions. :Erwinaze has been plagued by supply issues for years: reportedly, it can take 9 months to prepare (See, e.g., "Saving Ava: When a cancer drug shortage imperiled a toddler, her mom got to work", Stat News, Karen Weintraub (Oct. 31, 2016);
Asparaginase Erwinia Chrysanthemi Drug Shortage, Drugs.com). Even today the issues persist and Erwinase shortages are common. The FDA has issued warning letters to the manufacturer stating that "changes in the source material or cell line have a substantial potential to adversely affect the identity, strength, quality, purity or potency of Erwinase ."

[0005] There is a need for immunogenically non-cross reactive treatment options. A
L-asparaginase (like L-asparaginase recombinantly produced in Pseudomonas flumvscens) with no immunological cross-reactivity to E. coli-derived asparaginase would address a significant medical need (as a component of a multi-agent chemotherapeutic regimen) for patients with ALL/Lymphoblastic Lymphoma (LBL), by helping to ensure availability of an asparaginase for patients who have developed hypersensitivity to E. coli-derived asparaginase.
[0006] Currently, Erwinase is supplied as a sterile, lyophilized, white powder in vials. Each vial contains 10,000 International Units of asparaginase Erwinia chusanthemi, and the following inactive ingredients: glucose monohydrate (5.0 mg), sodium chloride (0.5 mg).
The availability of non-lyophilized formulations removes the need for rehydration before administration to a subject, e.g., a human. To that end, Oncaspar , an alternative, non-lyophilized treatment option, is supplied as a clear, colorless, preservative-free, isotonic sterile solution in phosphate-buffered saline, pH 7.3. Each milliliter contains 750 150 International Units of pegaspargase, dibasic sodium phosphate, USP (5.58 mg), monobasic sodium phosphate, USP, (1.20 mg) and sodium chloride, USP (8.50 mg) in water for injection, USP. In addition, ASPARLAS , another non-lyophilized alternative, is supplied as a clear, colorless, preservative-free, isotonic sterile solution in phosphate-buffered saline, pH
7.3, that requires dilution prior to intravenous infusion. Each vial of ASPARLAS contains 3,750 units in 5 mL
of solution. Each milliliter contains 750 units of calaspargase pegol-mknl;
dibasic sodium phosphate, USP (5.58 mg); monobasic sodium phosphate, USP (1.20 mg); and sodium chloride, USP (8.50 mg) in water for injection, USP. However, though non-lyophilized formulations (such as Oncaspar and ASPARLAS ) exist, there are issues with stability: for example, the existing formulations must be used within 48 hours if stored at room temperature.
[0007] Thus, there is a need in the art for non-lyophilized, stable, aqueous formulations of L-asparaginase.
BRIEF SUMMARY OF THE INVENTION
100081 Provided herein are aqueous, non-lyophilized formulations of L-asparaginase. In some aspects, such formulations may find use in the treatment of one or more diseases, disorders, or conditions treatable by asparagine depletion, including, for example, cancers such as Acute Lymphoblastic Leukemia (ALL) and Lymphoblastic Lymphoma (LBL), including relapsed ALL and relapsed LBL.

[0009] In one aspect, provided herein is an aqueous, non-lyophilized formulation, comprising:
(i) an L-asparaginase, wherein the L-asparanase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70%
identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
100101 In some 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, the one or more disaccharides comprise trehalose, sucrose, or any combination thereof In some embodiments, the one or more disaccharides comprise trehalose.
In some embodiments, the one or more buffers are substantially free of amino acid. In some embodiments, the one or more buffers comprise a phosphate buffer, an acetate buffer, or any combination thereof In some embodiments, the one or more buffers comprise a phosphate buffer. In some embodiments, the one or more buffers comprise sodium phosphate. In some embodiments, the sodium phosphate is sodium phosphate dibasic anhydrous, sodium phosphate monobasic monohydrate, or a combination thereof. In some embodiments, the formulation further comprises sodium chloride. In some embodiments, the formulation further comprises one or more excipients. In some embodiments, the one or more excipients comprise polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the L-asparaginase is present at a concentration of about 20 mg/triL. In some embodiments, the L-asparaginase is non-PECrylated and non-PASylated.
[0011] In some embodiments, the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 C for two months. In some embodiments, the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation comprises less than 2% high-molecular-weight (1.1MW) species after storage at 40 C for two months.
[0012] In one aspect, provided herein is an aqueous, non-lyophilized formulation, comprising:

(1) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: I;
(ii) one or more disaccharides, wherein the one or more di saccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 'V for one week.
[0013] In another aspect, provided herein is a method of treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject any of the formulations described herein.
[0014] In another aspect, provided herein is a unit dosage form, comprising: (i) any of the formulations described herein; and (ii) one or more pharmaceutically 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 that is treatable by asparagine depletion in a subject in need thereof.
DETAILED DESCRIPTION
DEFINITIONS
[0016] "Subject" refers to mammals, and includes humans and non-human mammals. Examples of individuals include, but are not limited to mice, rats, hamsters, guinea pigs, pigs, rabbits, cats, dogs, goats, sheep, cows, and humans. In some embodiments, subject refers to a human.
[0017] The articles "a" and "an" as used herein and in the appended claims are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, "a buffer"
means one buffer or more than one buffer.

4 100181 As used herein, "about" a parameter or value includes and describes that parameter or value per se. For example, "about X" includes and describes X per se. As used herein, the term "about" modifying, for example, the dimensions, volumes, quantity of an ingredient in a composition, concentrations, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term "about" the claims appended hereto include equivalents to these quantities. The term "about" further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term "about"
refers to a range of values that fall within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent or less of the stated reference value.
100191 As used herein, the terms "treatable by depletion of asparagine" and "treatable by asparagine depletion" refers to a disease, condition, or disorder wherein the cells involved in or responsible for the disease, condition, or disorder either lack or have a reduced ability to synthesize L-asparagine. Depletion or deprivation of L-asparagine can be partial or substantially complete (e.g., to levels that are undetectable using methods and apparatus that arc known in the art).
100201 The term "comprising the sequence of SEQ ID NO: 1"
means that the amino-acid sequence of the protein may not be strictly limited to SEQ ID NO: I
but may contain additional amino acids.
100211 "Low-molecular-weight species" or "LMW species", as used herein, refers to species that have a molecular weight that is lower than that of L-asparaginase. In some embodiments, the LMW species have a molecular weight that is less than about 100%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the molecular weight of L-asparaginase. In some embodiments, LMW species are formed by the degradation or decomposition of L-asparaginase, e.g., through hydrolysis of one or more covalent bonds in the L-asparaginase.
100221 -High-molecular-weight species" or "HMW species", as used herein, refers to species that have a molecular weight that is greater than that of L-asparaginase. In some embodiments, the HMW species have a molecular weight that is greater than about 100%, greater than about 150%, greater than about 200%, greater than about 250%. greater than about 300%, greater than about 350f/o, greater than about 400%, greater than about 450%, or greater than about 500% of the molecular weight of L-asparaginase.
10023.1 "Trehalose" refers to a disaccharide having the following structure:
HO
OH
HOõ,. .s.cH0,,.....).,........,,OH 2 .__,...,=,10's ..õ.õ
HO
OH
' 100241 "Sucrose" refers to a disaccharide having the following structure:
HO,,, _ 0õ,",.....OH
HO.,..õ).¨c--J,õ
06- 1:11-1 HO
He. ''OH
OH
=
100251 "Polysorbate 80" or "PS-80" refers to an excipient having the following structure:

w0 HOÃ--c) w+x+y+z=20 100261 "PEGyiated", 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 way (e.g., through one or more covalent bonds) and at any suitable location of the structure or moiety in question. For example, a "PEGylated L-asparaginase" indicates an L-asparaginase to which one or more PEG units have been conjugated or attached. Accordingly, a "non-PEGylated" moiety is one to which PEG has not been conjugated or attached.
100271 "PA Sylation", as used herein, is used to describe a structure or moiety to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, is conjugated attached. Any suitable number of such polypeptide units may be attached in any suitable way (e.g., through one or more covalent bonds) and at any suitable location of the structure or moiety in question. For example, a "PASylated L-asparaginase" indicates an. L-asparaginase to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, have been conjugated or attached.
Accordingly, a "non-PASylated" moiety is one to which one or more polypeptide units comprising proline, alanine, or serine, or any combination thereof, has not been conjugated or attached.
100281 "SEQ ID NO: 1" is as follows:
ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQF
SNMASENMTGDVVLKLSQRVN. ELLARDDVDGVVITHGTDTVEESAYFLHLTVKSD
KPVVFVAAMRPATAISADGPMNLLEAVRVAGUKQSRGR.GVIVIVVLNDRIGSARYIT
KTNASTLDTFKANEEGYLGVIIGNRIYYQN'TUDKLHTTRSVFDVRGLTSLPKVDILYG
YQ.DD.PEYLYDAAIQII.GVKGIVYAGMGAGSVSV.RGIAGMRICAMEKGVVVIRSTRTG
NGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY.

FORMULATIONS
100291 In one aspect, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70%
identical to SEQ ID NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.
100301 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 sorbitols, 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 butlers. In some embodiments, the one or more buffers, wherein the one or more buffers are substantially free of amino acid. In some embodiments, the L-asparaginase formulation comprises one or more stabilizers and one or more buffers. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 'C for two months. In some embodiments, the L-asparaginase formulation comprises less than 2% high-molecular-weight (11MW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species and less than 2% high-molecular-weight (I-EVIW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 C for one week.
100311 In one aspect, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an I .-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95%
identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week.

100321 In one aspect, a L-asparaginase in accordance with the disclosure provided herein is an recombinant L-asparaginase. In a further aspect, a L-asparaginase in accordance with the invention described herein is an enzyme with L-asparagine aminohydrolase activity.
Such a L-asparaginase's enzymatic activity may include not only deamidation of asparagine to aspartic acid and ammonia, but also deamidation of glutamine to glutamic acid and ammonia.
100331 In some embodiments, a L-asparaginase as disclosed herein is active as a multimer. In some embodiments, the L-asparaginase is an active enzyme as a tetramer. A
tetramer is composed of four subunits (also known as monomers). In some embodiments, a L-asparaginase is a tetramer consisting of four identical 35 kD subunits. In some embodiments, the L-asparaginase is a non-disulfide bonded tetrameric therapeutic protein. In a particular embodiment, each of the subunits or monomers of a multimeric L-asparaginase comprises the amino acid sequence of S:EQ113 NO: 1.
100341 In a particular embodiment, each of the subunits or monomers of a tetrameric Lasparaginase comprises the amino acid sequence of SEQ 1:13 NO: 1.
In another embodiment, the L-asparaginase is from Erwinia chrysanthemi NCPPB 1066 (Genbank Accession No. CAA32884, incorporated herein by reference in its entirety), either with or without signal peptides and/or leader sequences.
100351 In some embodiments, the L-asparaginase is composed of multiple subunits, for example, four subunits or monomers (tetramer). A corresponding modified protein may then, e.g., consist of 1 to 20 (or more) peptides conjugated to each of the monomers of that tetramer. 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 L-asparaginase monomers. In a specific embodiment, the L-asparaginase is a multimer comprising multiple subunits or monomers, such as a tetramer, and each of the monomers in that tetramer is conjugated to 1 peptide, resulting in a tetramer comprising 4 conjugated peptides, one for each monomer. In some embodiments, the L-asparaginase is a tetramer comprising 1-4 peptides conjugated to each of the monomers.
100361 In some embodiments, the L-asparaginase is a tetramer comprising 4-20 peptides conjugated to each of the L- monomers. In some embodiments, the Lasparaginase is a tetramer comprising 6-18 peptides conjugated to each of the monomers. In some embodiments, the L-asparaginase is a tetramer comprising 6-18 peptides conjugated to each of the monomers. In some embodiments, the L-asparaginase is a tetramer comprising 10-15 peptides conjugated to each of the monomers.
100371 In one aspect, the invention relates to a modified protein having a L-asparaginase and multiple chemically attached peptide sequences. In a further aspect the length of the peptide sequences are from about 10 to about 100, from about 15 to about 60 or from about 20 to about 40.
100381 Fragments of L-asparaginase, preferably fragments of the L-asparaginase of SEQ ID NO: 1, may be of use in the presently described invention. The term "a fragment of L-asparaginase" (e.g., a fragment of the L-asparaginase of SEQ ID NO: 1) means that the sequence of the L-asparaginase may include fewer amino-acids than in the L-asparaginases exemplified herein (e.g., the L-asparaginase of SEQ ID NO: 1) but still enough amino-acids to confer L-aminohydrolase activity. For example, a "fragment of L-asparaginase" is a fragment that is/consists of at least about 150 or 200 contiguous amino acids of one of the asparaginases exemplified herein (e.g. the L-asparaginase of SEQ ID NO: 1) (for example, about 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 321, 322, 323, 324, 325, or 326 contiguous amino acids) and/or wherein said fragment has up to 50 amino acids deleted from the N-terminus of said L-asparaginases exemplified herein (e.g. the L-asparaginase of SEQ ID NO: 1) (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50) and/or has up to up to 75 or 100 amino acids deleted from the C-terminus of said L-asparaginases exemplified herein (e.g., the Lasparaginase of SEQ ID NO:
1) (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) and/or has deleted amino acids at both the N-terminus and the C-terminus of said L-asparaginases exemplified herein (e.g., the L-asparaginase of SEQ ID
NO: 1), wherein the total number of amino acids deleted can be up to 125 or 150 amino acids.
100391 Indeed, a person skilled in the art will understand how to select and design homologous proteins retaining substantially their L-asparaginase activity.
Typically, a Nessler assay is used for the determination of L-asparaginase activity according to a method described by Mashburn and Wriston (Mashburn, L., and Wriston, J. (1963) "Tumor Inhibitory Effect ofLAsparaginase," Biochem Biophys Res Commun 12, 50, incorporated herein by reference in its entirety).

100401 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. The term "one or more amino acids" in this context can refer to one, two, three, four, five, six, seven, eight, nine, ten or more amino acids. For example, substitution of one amino-acid at a given position by a chemically equivalent amino-acid that does not affect the functional properties of a protein is common Substitutions may be defined as exchanges within one of the following groups: Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin;
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 negatively charged residue for another (such as glutamic acid for aspartic acid) or one positively charged residue for another (such as lysine for arginine) can be expected to produce a functionally equivalent product.
[0042] The positions where the amino-acids are modified and the number of amino-acids that may be modified in the amino-acid sequence are not particularly limited.
The skilled artisan is able to recognize the modifications that can be introduced without affecting the activity of the protein. For example, modifications in the N- or C-terminal portion of a protein may be expected not to alter the activity of a protein under certain circumstances. With respect to asparaginases, in particular, much characterization has been done, particularly with respect to the sequences, structures, and the residues forming the active catalytic site. This provides guidance with respect to residues that can be modified without affecting the activity of the enzyme. All known Lasparaginases from bacterial sources have common structural features. 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 sequences of the catalytic sites off--asparaginases are highly conserved between Erwinia chrysanthemi, Erwinia carotovora, and E. call L-asparaginase II (Id). The active site flexible loop contains amino acid residues 14-33, and structural analysis show that ThrI5, Thr95, Ser62, Glu63, Asp96, and AlaI20 contact the ligand (Id). Aghaipour et al. have conducted a detailed analysis of the four active sites of Erwinia chlysanthemi L-asparaginase by examining high resolution crystal structures of the enzyme complexed with its substrates (Aghaipour (2001) Biochemistry 40, 5655-5664). Kotzia et al. provide sequences for L-a.sparaginases from several species and subspecies of Erwinia and, even though the proteins have only about 75-77% identity between Erwinia chrysanthemi and Erwinia carotovora, they each still have L-asparaginase activity (Kotzia (2007) J. Biotechnol. 127, 657-669). Moola et al performed epitope mapping studies of Erwinia chtysanthemi 3937 L-asparaginase and were able to retain enzyme activity even after mutating various antigenic sequences in an attempt to reduce immunogenicity of the asparaginase (Moola (1994) I3iochem. J. 302, 921-927). In view of the extensive characterization that has been performed on L-asparaginases, one of skill in the art could determine how to make fragments and/or sequence substitutions while still retaining enzyme activity.
100431 :More specifically, fragments of the protein of S:EQ
ID NO: 1 are also comprised within the definition of the protein used in the L-asparaginase of the invention.
The term "a fragment of SEQ ID NO: 1" means that the sequence of the polypeptide may include fewer amino-acids than the full-length SEQ ID NO: 1 but retains enough of the protein to confer Laminohydrolase activity. In some embodiments, a L-asparaginase has at least about 80% homology or identity with the protein comprising SEQ ID NO: 1.
100441 In some embodiments, the L-asparaginase of the formulation comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1. In some embodiments, each monomer unit has an amino acid sequences that is at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, or about 84% identical to SEQ ID NO: 1. In some embodiments, each monomer unit has an amino acid sequences that is at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90% about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID
NO: I.
100451 in some embodiments, the L-asparaginase of the formulation is present at a concentration of about 20 mg/mL.
100461 In some embodiments, the L-asparaginase of the formulation is non-PECrylated. In some embodiments, the L-asparaginase of the formulation is non-PASylated.

In some embodiments, the L-asparaginase of the formulation is non-PEGylated and non-PASylated.

In some embodiments, in conjunction with embodiments above or below, the L-asparaginase formulation comprises one or more stabilizers. In some embodiments, in conjunction with embodiments above or below, 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 embodiments above or below, the L-asparaginase formulation comprises one or more buffers. In some embodiments, in conjunction with embodiments above or below, the one or more buffers comprise acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane (e.g., Tris), or any combination thererof. In some embodiments, in conjunction with embodiments above or below, the L-asparaginase formulation comprises one or more surfactants. In some embodiments, in conjunction with embodiments above or below, 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 embodiments above or below, the L-asparaginase formulation comprises one or more polyol (e.g., mannitol and sorbitol), one or more di saccharides (e.g., sucrose and trehalose), and one or more polysaccharides (e.g., dextran 40). In some embodiments, in conjunction with embodiments above or below, the L-asparaginase formulation comprises sodium chloride. In some embodiments, in conjunction with embodiments above or below, the L-asparaginase formulation comprises one or more amino acids (e.g., proline, glycine, and arginie).

In some embodiments, in conjunction with embodiments above or below, the formulation comprises one or more stabilizers at a concentration of between about 50 mM
and about 300 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 75 mM and about 300 mM, between about 100 m:M and about 300 mM, between about 125 mM and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM and about 300 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 50 mM
and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM
and about 225 mM, between about 50 naM and about 200 mM, between about 50 mM: and about 175 mM, between about 50 mM and about 150 mM, between about 50 mM and about mM, between about 50 mM and about 100 mM, or between about 50 rn.114 and about 75 mM.
In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 150 mM and about 275 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 150 mM
and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM and about 180 mM, between 150 mM and about 170 m:M, or between about 160 mM and 150 mM. In some embodiments, the formulation comprises one or more stabilizers at a concentration of between about 160 mM and about 200 m:M, between about 170 mM: and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM.
In some embodiments, the formulation comprises one or more stabilizers at a concentration of about 170 mM.
100491 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.
100501 In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 50 mM and about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 75 mM and about 300 mM, between about 100 mM and about 300 mM, between about 125 mM and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM and about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 50 mM
and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM and about mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 tn_M, between about 50 mM and about 150 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mM, or between about 50 mM and about 75 mM.
In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 150 mM and about 275 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 150 mM
and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM and about mM, between 150 mM and about 170 mM, or between about 160 mM and 150 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 160 mM and about 200 mM, between about 170 mM and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM.
In some embodiments, the formulation comprises one or more disaccharides at a concentration of about 170 mM.
00511 In som.e embodiments, the formulation comprises trehalose at a concentration of between about 50 mM and about 300 mM. In some embodiments, the formulation comprises one or more disaccharides at a concentration of between about 75 mM
and about 300 mM, between about 100 mM and about 300 mM, between about 125 mM
and about 300 mM, between about 150 mM and about 300 mM, between about 175 mM and about 300 mM, between about 200 mM and about 300 mM, between about 225 mM and about 300 mM, between about 250 mM and about 300 mM, or between about 275 mM
and about 300 mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 50 mM and about 275 mM, between about 50 mM and about 250 mM, between about 50 mM and about 225 mM, between about 50 mM and about mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 50 mM and about 125 mM, between about 50 mM and about 100 mivl, or between about 50 mM and about 75 mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 150 mM and about 275 mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 150 /TIM and about 200 mM, between about 150 mM and about 190 mM, between about 150 mM
and about 180 mM, between 150 mM and about 170 mM, or between about 160 mM and mM. In some embodiments, the formulation comprises trehalose at a concentration of between about 160 mM and about 200 mM, between about 170 mM and about 200 mM, between about 180 mM and about 200 mM, or between about 190 mM and about 200 mM.
In some embodiments, the formulation comprises trehalose at a concentration of about 170 mM.
100521 In some embodiments, the one or more buffers of the formulation are substantially free of amino acid. In some embodiments, the one or more buffers of the formulation are substantially free of histidine or arginine, or both. In some embodiments, the one or more buffers of the formulation are substantially free of histidine. In some embodiments, the one or more buffers of the formulation comprise a phosphate buffer, an acetate buffer, or any combination thereof. In some embodiments, the one or more buffers comprise a phosphate buffer. In some embodiments, the one or more buffers comprise an acetate buffer. In some embodiments, the one or more buffers comprise a combination of a phosphate buffer and an acetate buffer. In some embodiments, the one or more buffers comprise an acetate salt of an alkali metal. In some embodiments, the one or more buffers comprise a phosphate salt of an alkali metal. In some embodiments, the one or more buffers comprise sodium phosphate.
[00531 In some embodiments, formulation comprises one or more buffers at a concentration of between about 0.5 mM and between about 50 mM. In some embodiments, formulation comprises one or more buffers at a concentration of between about 1 mM and about 50 mM, between about 2.5 m M and about 50 mM, between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 mM, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM. In some embodiments, formulation comprises one or more buffers at a concentration of between about 0.5 mM and about 45 m1\4, between about 0.5 mM and about 40 mM, between about 0.5 mM and about 35 mM, between about 0.5 mM and about 30 mM, between about 0.5 mM and 25 mM, between about 0.5 mM and about 20 mM, between about 0.5 mM and about 15 mM, between about 0.5 mM and about 10 mM, between about 0.5 mM and about 5 mM, between about 0.5 M
and about 2.5 mM, or between 0.5 mM and about 1 mM. In some embodiments, formulation comprises one or more buffers at a concentration of between about 10 mM and about 30 mM.
In some embodiments, formulation comprises one or more buffers at a concentration of between about 15 mM and about 25 mM. In some embodiments, formulation comprises one or more buffers at a concentration of about 20 mM.
100541 In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 0.5 mM and between about 50 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 1 mM and about 50 mM, between about 2.5 mM and about 50 mM, between about 5 mM and about 50 mM, between about 10 mM and about 50 mM, between about 15 mM and about 50 mM, between about 20 mM and about 50 mM, between about 25 mM and about 50 mM, between about 30 mM and about 50 mM, between about 35 mM and about 50 m114, between about 40 mM and about 50 mM, or between about 45 mM and about 50 mM. in some embodiments, the formulation comprises sodium phosphate at a concentration of between about 0.5 mM and about 45 mM, between about 0.5 mM and about 40 mM, between about 0.5 rriM and about 35 mM, between about 0.5 mM and about 30 mM, between about 0.5 mM and 25 mM., between about 0.5 mM and about 20 mM, between about 0.5 mM and about 15 mM, between about 0.5 mM and about 10 mM, between about 0.5 mM and about 5 mM, between about 0.5 M
and about 2.5 mM, or between 0.5 mM and about 1mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 10 mM and about 30 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of between about 15 mM and about 25 mM. In some embodiments, the formulation comprises sodium phosphate at a concentration of about 20 mM.

In some embodiments, the formulation further comprises an alkali metal salt. In some embodiments, the formulation further comprises a halide salt. In some embodiments, the formulation further comprises a halide salt of an alkali metal. In some embodiments, the formulation further comprises sodium chloride. In some embodiments, the formulation further comprises sodium chloride at a concentration of between about 25 mM
and about 150 mM. In some embodiments, the formulation further comprises sodium chloride at a concentration of between about 30 mM and about 150 mM, between about 40 mM and about 150 mM, between about 50 mM and about 150 mM, between about 60 mM.
and about 150 mM, between about 70 mM and about 150 m:M, between about 80 mM
and about 150 mM, between about 90 mM and about 150 mM, between about 100 mM and about 150 mM, between about 110 mM and about 150 mM, between about 120 mM and about mM, between about 130 mM and about 150 mM, or between about 140 mM and about mM. In some embodiments, the formulation comprises sodium chloride at a concentration of between about 25 mM and about 140 mM, between about 25 mM and 130 mM, between about 25 mM and about 120 mM, between about 25 mM and about 110 mM, between about 25 mM and about 100 mM, between about 25 mM and about 90 mM, between about 25 mM
and about 80 mM, between about 25 mM and about 70 mM, between about 25 mM and about 60 mM, between about 25 mM and about 50 mM, between about 25 mM and about 40 mM, or between about 25 mM and 30 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of between about 30 mM and about 70 rnM. In some embodiments, the formulation comprises sodium chloride at a concentration of between about 40 mM and about 60 mM. In some embodiments, the formulation comprises sodium chloride at a concentration of about 50 mM.
100561 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.
100571 In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.3% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.28% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.26% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.24% (w/v). In some embodiments, the formulation comprises one or more excipients at a concentration of no more than about 0.22% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.3% (w/v).
In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.3% (w/v).
100581 In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.2%
(w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.004% (w/v) and about 0.18% (w/v), between about 0.004% (w/v) and about 0.16% (w/v), between about 0.004% (w/v) and about 0.14% (w/v), between about 0.004%
(w/v) and about 0.12% (w/v), between about 0.004% (w/v) and about 0.1% (w/v), between about 0.004% (w/v) and about 0.08% (w/v), between about 0.004% (w/v) and about 0.06%
(w/v), between about 0.004% (w/v) and about 0.04% (w/v), between about 0.004%
(w/v) and about 0.03%, between about 0.004% (w/v) and about 0.025% (w/v), between about 0.004%
(w/v) and about 0.02% (w/v), between about 0.004% (w/v) and about 0.015%
(w/v), or between about 0.004% (w/v) and about 0.01% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.2% (w/v), between about 0.015% (w/v) and about 0.2% (w/v), between about 0.02% (w/v) and about 0.2% (w/v), between about 0.025% (w/v) and about 0.2%
(w/v), between about 0.03% (w/v) and about 0.2% (w/v), between about 0.04% (w/v) and about 0.2% (w/v), between about 0.05% (w/v) and about 0.2% (w/v), between about 0.06% (w/v) and about 0.2% (w/v), between about 0.07% (w/v) and about 0.2% (w/v), between about 0.08% (w/v) and about 0.2% (w/v), between about 0.09% (w/v) and about 0.2%
(w/v), between about 0.1% (w/v) and about 0.2% (w/v), between about 0.12% (w/v) and about 0.2%
(w/v), between about 0.14% (w/v) and about 0.2% (w/v), between about 0.16%
(w/v) and about 0.2% (w/v), or between about 0.18% (w/v) and 0.2% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.01% (w/v) and about 0.03% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of between about 0.015%
(w/v) and about 0.025% (w/v). In some embodiments, the formulation further comprises one or more excipients at a concentration of about 0.02% (w/v).
100591 In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.004% (w/v) and about 0.2% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.004% (w/v) and about 0.18% (w/v), between about 0.004% (w/v) and about 0.16%
(w/v), between about 0.004% (w/v) and about 0.14% (w/v), between about 0.004%
(w/v) and about 0.12% (w/v), between about 0.004% (w/v) and about 0.1% (w/v), between about 0.004% (w/v) and about 0.08% (w/v), between about 0.004% (w/v) and about 0.06%
(w/v), between about 0.004% (w/v) and about 0.04% (Aviv), between about 0.004% (w/v) and about 0.03%, between about 0.004% (w/v) and about 0.025% (w/v), between about 0.004%
(w/v) and about 0.02% (w/v), between about 0.004% (w/v) and about 0.015% (w/v), or between about 0.004% (w/v) and about 0.01% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.01% (w/v) and about 0.2%
(w/v), between about 0.015% (w/v) and about 0.2% (w/v), between about 0.02%
(w/v) and about 0.2% (w/v), between about 0.025% (w/v) and about 0.2% (w/v), between about 0.03%
(w/v) and about 0.2% (w/v), between about 0.04% (w/v) and about 0.2% (w/v), between about 0.05% (w/v) and about 0.2% (w/v), between about 0.06% (w/v) and about 0.2% (w/v), between about 0.07% (w/v) and about 0.2% (w/v), between about 0.08% (w/v) and about 0.2% (w/v), between about 0.09% (w/v) and about 0.2% (w/v), between about 0.1%
(w/v) and about 0.2% (w/v), between about 0.12% (w/v) and about 0.2% (w/v), between about 0.14% (w/v) and about 0.2% (w/v), between about 0.16% (w/v) and about 0.2%
(w/v), or between about 0.18% (w/v) and 0.2% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.01% (w/v) and about 0.03%
(w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of between about 0.015% (w/v) and about 0.025% (w/v). In some embodiments, the formulation further comprises polysorbate 80 at a concentration of about 0.02% (w/v).
[00601 In some embodiments, the formulation provided herein has a pI1 of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH
of between about 4.5 and about 8.5, between about 5.0 and about 8.5, between about 5.5 and about 8.5, between about 6.0 and about 8.5, between about 6.5 and about 8.5, between about 7.0 and about 8.5, between about 7.5 and about 8.5, or between about 8.0 and about 8.5. In some embodiments, the formulation has a pH of between about 4.5 and about 8.0, between about 4.5 and about 7.5, between about 4.5 and about 7.0, between about 4.5 and about 6.5, between about 4.5 and about 6.0, between about 4.5 and about 5.5, or between about 4.5 and about 5Ø In some embodiments, the formulation provided herein has a pH of between about 4.0 and about 8.0, between about 4.0 and about 7.5, between about 4.0 and about 7.0, between about 4.0 and about 6.5, between about 4.0 and about 6.0, between about 4.0 and about 5.5, between about 4.0 and about 5.0, or between about 4.0 and about 4.5. In some embodiments, the formulation provided herein has a pH of between about 5.5 and about 8.5.
In some embodiments, the formulation provided herein has a pH of between about 6.0 and about 8Ø In some embodiments, the formulation provided herein has a pH of between 6.5 and about 7.5. In some embodiments, the formulation provided herein has a pH
of around 7Ø
[00611 In some embodiments, in conjunction with embodiments above or below, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO:
1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof. 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 sorbitols, 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-sparaginase formulation comprises one or more buffers.
In some embodiments, the one or more buffers, wherein the one or more buffers are substantially free of amino acid. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than 2% high-molecular-weight (1-LMW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (UMW) species and less than 2% high-molecular-weight (HMW) species after storage at 40 'C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5.
In some embodiments, the formulation has a pH of about 7Ø

In some embodiments, in conjunction with embodiments above or below, (i) the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and is present at a concentration of around 20 mg/mL; (ii) one or more stabilizers (e.g., disaccharides, sorbitols, amino acids, or any combination thereof) at a concentration between about 50 m:M and about 300 mM; (iii) one or more buffers (e.g., acetate, glutamate, citrate, histidine, succinate, phosphate, hydroxymethylaminomethane, or combination thereof) at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM; and (v) one or more surfactants (e.g., polysotbate 80, polysorbate 20, poloxamer 188, or any combination thereof) at a concentration of between about 0.004% (w/v) and about 0.3%
(w/v). In some embodiments, in conjunction with embodiments above or below, the formulation has a p1-I of between about 4.0 and about 8.5. In some embodiments, in conjunction with embodiments above or below, the formulation has a pH of about 7Ø

In some embodiments, in conjunction with embodiments above or below, (i) the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO: 1, and is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration between about 50 mM and about 300 mM; (iii) sodium phosphate, wherein the sodium phosphate (e.g., dibasic anhydrous, monobasic monohydrate, or combination thereof) is present at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM
and about 150 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of between about 0.004% (w/v) and about 0.2% (w/v).
100641 In some embodiments, in conjunctions with embodiments above or below, the L-asparaginase can be formulated in a unit dosage form. The term "unit dosage forms"
refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. In some embodiments, a unit dosage can be administered every 12 hours, every 24 hours, every 48 hours, or every 72 hours. In some embodiments, a unit dosage is administered every 48 hours. In some embodiments, a unit dosage contain from about 5 mg to about 50 mg (e.g., about 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 fig, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, and 49 mg) of the L-asparaginase.
100651 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more di saccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid;
and (iv) sodium chloride, wherein the formulation comprises less than about 5% low-molecular-weight (I.,M'W) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø

100661 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid;
and (iv) one or more excipients, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week.
100671 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more di saccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid;
(iv) sodium chloride; and (v) one or more excipients, wherein the formulation comprises less than about

5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø
100681 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid;
and (iv) polysorbate 80, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø
100691 in some embodiments, provided herein is an aqueous, non-lyophilized formulafion, comprising: (1) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid;
(iv) sodium chloride; and (v) polysorbate 80, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø
100701 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1; (ii) trehalose; (iii) sodium phosphate; (iv) sodium chloride;
and (v) polysorbate 80, wherein the formulation comprises less than about 5%
low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø
100711 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration between about 50 mM and about 300 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of between about 0.5 mM and about 50 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of between about 0.004% (w/v) and about 0.2% (w/v), wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C
for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø
100721 In some embodiments, provided herein is an aqueous, non-lyophilized formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1, and wherein the L-asparaginase is present at a concentration of around 20 mWrnL; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM; and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v), wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 "C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5.
In some embodiments, the formulation has a pH of about 7Ø
100731 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 storage at 37 "C for one week. In. some embodiments, the formulation comprises less than about 4.5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation comprises less than about 4% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation comprises less than about 3.5% low-molecular-weight (LMW) species after storage at 37 'C for one week. In some embodiments, the formulation comprises less than about 3% low-molecular-weight (LMW) species after storage at 37 C
for one week. In some embodiments, the formulation comprises less than about 2.5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation comprises less than about 2% low-molecular-weight (LMW) species after storage at 37 C for one week. the formulation comprises less than about 1.5%
low-molecular-weight (LMW) species after storage at 37 "C for one week. In some embodiments, the formulation comprises less than about 1% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation comprises less than about 0.5% low-molecular-weight (LMW) species after storage at 37 "C
for one week.
100741 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, the same as if each and every combination of the components and/or features described herein were individually and specifically listed.

MET:HODS OF TREATMENT
1007m The L-asparaginase formulations of the present disclosure can be used in the treatment of a disease in a subject (for example, a human), wherein the disease is treatable by the depletion of asparagine or the administration of asparaginase. In some embodiments, the human subject has, prior to administration of the L-asparaginase, experienced silent inactivation of the E. Coli-derived asparaginase. In some embodiments, the subject has, prior to administration of the L-asparaginase, experienced an allergic reaction to the E. Coli-derived asparaginase. In some embodiments, the subject has, prior to administration of the L-asparaginase, experienced anaphylaxis to the E. Coli-derived asparaginase. Non-limiting examples of objective signs of allergy or hypersensitivity include testing "antibody positive"
for an asparaginase enzyme.
100761 In some embodiments, the L-asparaginase formulations of the present disclosure are useful in the treatment or the manufacture of a medicament for use in the treatment of acute lymphoblastic leukemia (ALL). The incidence of relapse in ALL patients following treatment with L-asparaginase remains high, with approximately 10-25% of pediatric ALL patients having early relapse (e.g., some during maintenance phase at 30-36 months post-induction). If a patient treated with E. coli-derived L-asparaginase has a relapse, subsequent treatment with E coli preparations could lead to a "vaccination"
effect, whereby the E. coil preparation has increased immunogenicity during the subsequent administrations.
In one embodiment, the L-asparaginase of the invention may be used in a method of treating patients with relapsed ALL who were previously treated with other asparaginase preparations, in particular those who were previously treated with E. coli-derived asparaginases.
100771 :Diseases or disorders that the L-asparaginase formulations of the present disclosure are useful in treating include, but are not limited to, the following: malignancies, or cancers, including but not limited to, hematalogic malignancies, lymphoma, non-Hodgkin's lymphoma, NK lymphoma, pancreatic cancer, Hodgkin's disease, large cell immunoblastic lymphoma, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute T-cell leukemia, acute myeloid leukemia (AML), biphenotypic 13-cell myelomonocytic Leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma, and diffuse large B-cell lymphoma (DLBCL). Other diseases or disorders that the L-asparaginase formulations useful in treating are cancers including, but not limited to, renal cell carcinoma, renal cell adenocarcinoma, glioblastoma including glioblastoma multiforma and glioblastoma astrocytoma, medulloblastoma, rhabdomyosarcoma, malignant melanoma, epidermoid carcinoma, squamous cell carcinoma, lung carcinoma including large cell lung carcinoma and small cell lung carcinoma, endometrial carcinoma, ovarian adenocarcinoma, ovarian tetratocarcinoma, cervical adenocarcinoma, breast carcinoma, breast adenocarcinoma, breast ductal carcinoma, pancreatic adenocarcinoma, pancreatic ductal carcinoma, colon carcinoma, colon adenocarcinoma, colorectal adenocarcinoma, bladder transitional cell carcinoma, bladder papilloma, prostate carcinoma, osteosarcoma, epitheloid carcinoma of the bone, prostate carcinoma, and thyroid cancer. The cancer may be a solid cancer, for example lung cancer or breast cancer. Representative non-malignant hematologic diseases which respond to asparagine depletion include immune system-mediated blood diseases, including, but not limited to, infectious diseases such as those caused by HIV infection (i.e., AIDS) Nonhematologic diseases associated with asparagine dependence include autoimmune diseases, for example, rheumatoid arthritis, collagen vascular diseases, AIDS, osteoarthritis, Issac's syndrome, psoriasis, insulin dependent diabetes mellitus, multiple sclerosis, sclerosing panencephalitis, systemic lupus erythematosus (SLE), rheumatic fever, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), primary billiary cirrhosis, chronic active hepatitis, glomerulonephritis, myasthenia gravis, pemphigus vulgaris, and Graves' disease.
Cells suspected of causing disease can be tested for asparagine dependence in any suitable in vitro or in vivo assay, e.g., an in vitro assay wherein the growth medium lacks asparagine.

In some embodiments, in conjunction with embodiments above or below, the disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof comprises AVNT mutated colorectal cancer (CRC). In some embodiments, in conjunction with embodiments above or below, the disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof comprises relapse remitting (R/R) acute myeloid leukemia (AML).

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

Pharmaceutical compositions and co-administration 100801 In some embodiments, treatment with a L-asparaginase formulation of the present disclosure is co-administered with a multi-agent chemotherapeutic regimen. In some embodiments, treatment with a L-asparaginase formulation of the present disclosure is co-administered with one or more other chemotherapeutic agents as part of a multi-agent chemotherapeutic regimen. In some embodiments, treating patients with a L-asparaginase formulation of the present disclosure in addition to other agents helps to ensure availability of an asparaginase for patients who have developed hypersensitivity to K coil derivedasparaginase.
100811 Examples of agents that may be part of a multi-agent chemotherapeutic regimen with a L-asparaginase of the present disclosure include, but are not limited to:
cytarabine, vincristine, daunorubicin, methotrexate, leuvocorin, doxorubicin, anthracycline, corticosteroids and glucocortiods (including but not limited to prednisone, prednisolone, and/or dexamethasone), cyclophosphamide, 6-mercaptopurine, venetoclax, and etoposi de. In some embodiments, the multi-agent chemotherapeutic regimen is the L-asparaginase and one additional chemotherapeutic agent. In some embodiments, the multi-agent chemotherapeutic regimen is the L-asparaginase and two or more additional chemotherapeutic agents.
100821 As an example, patients with ALL will be co-administered the L-asparaginase of the present disclosure along with a multi-agent chemotherapy during 3 chemotherapy phases including induction, consolidation or intensification, and maintenance.
In a specific example, the L-asparaginase formulation of the present disclosure is co-administered with an asparagine synthetase inhibitor (e.g., such as set forth in WO
2007/103290, which is herein incorporated by reference in its entirety). 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 drugs. In another specific example, the L-asparaginase formulation of the present disclosure is co-administered with an asparagine synthetase inhibitor and other chemotherapy drugs. The L-asparaginase formulation of the present disclosure can be co-administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen. in a particular embodiment, the I,-asparaginase of the present disclosure comprises a protein recombinantly produced in Pseudomonasfluorescens and, more specifically, the L-asparaginase comprising the sequence of SEQ ID NO: 1.

100831 The formulations described herein can be administered to a patient as a pharmaceutical composition using standard techniques. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, 22nd edition, Mack Publishing, 2015 (herein incorporated by reference).
100841 Suitable dosage forms, in part, depend upon the use or the route of entry, for example, oral, transdemial, transmucosal, or by injection (parenteral).
Such dosage forms should allow the therapeutic agent to reach a target cell or otherwise have the desired therapeutic effect. For example, pharmaceutical compositions injected into the blood stream preferably are soluble. The pharmaceutical compositions according to the present disclosure can be formulated as pharmaceutically acceptable salts and complexes thereof Pharmaceutically acceptable salts are non-toxic salts present in the amounts and concentrations at which they are administered The preparation of such salts can facilitate pharmaceutical use by altering the physical characteristics of the compound without preventing it from exerting its physiological effect. Useful alterations in physical properties include lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate administering higher concentrations of the drug. The pharmaceutically acceptable salt of a modified protein as described herein may be present as a complex, as those in the art will appreciate. Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, fumarate, maleate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate, and quinate. Pharmaceutically acceptable salts can be obtained from acids, including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, fumaric acid, and quinic acid. Pharmaceutically acceptable salts also include basic addition salts such as those containing benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium, ammonium, alkylamine, and zinc, when acidic functional groups, such as carboxylic acid or phenol are present. For example, see Remington's Pharmaceutical Sciences, supra.
Such salts can be prepared using the appropriate corresponding bases.
Pharmaceutically acceptable carriers and/or excipients can also be incorporated into a pharmaceutical composition according to the invention to facilitate administration of the particular asparaginase. Examples of carriers suitable for use in the practice of the 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 solution and dextrose.
100851 Pharmaceutical compositions according to the invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, topical (transdermal), or transmucosal administration.
For oral administration, for example, the compounds can be formulated into conventional oral dosage forms such as capsules, tablets, and liquid preparations such as syrups, elixirs, and concentrated drops. Alternatively, injection (parenteral administration) may be used, e.g., intramuscular, intravenous, intraperitoneal, and subcutaneous injection. For injection, pharmaceutical compositions are formulated in liquid solutions, preferably in physiologically compatible buffers or solutions, such as saline solution, Hank's solution, or Ringer's solution.
In a specific aspect, the L-asparaginase formulation is administered intramuscularly. In a specific aspect, the L-asparaginase formulation is administered intravenously.
In a specific aspect, the L-asparaginase formulation is administered subcutaneously.
100861 Systemic administration can also be accomplished by transmucosal or transderrnal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are well known in the art, and include, for example, for transmucosal administration, bile salts, and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation.
Transmucosal administration, for example, may be through nasal sprays, inhalers (for pulmonary delivery), rectal suppositories, or vaginal suppositories. For topical administration, compounds can be formulated into ointments, salves, gels, or creams, as is well known in the art.
Dosing 100871 In some embodiments, a dose is an amount administered to the human subject over a certain time and frequency. In some embodiments, the dose of L-asparaginase formulation will be given to a human subject with hypersensitivity only when the hypersensitivity subsides.
100881 In an exemplary embodiment, al--asparaginase formulation is administered to a human subject in an amount from about 10 mg/m2 to 100 mg/m2.
In an exemplary embodiment, a L-asparaginase formulation is administered intramuscularly every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 25 mg/m2. In an exemplary embodiment, a L-asparaginase formulation is administered intravenously every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 37.5 mg/m2. In an exemplary embodiment, a L-asparaginase formulation is administered intravenously every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, wherein the amount is about 50 mg/m2.
100891 In further exemplary embodiments and in accordance with any of the discussion herein regarding dosing, the L-asparaginase formulation administered in such doses is not conjugated to a polymer such as a PEG moiety and/or is not conjugated to a peptide comprising solely alanine and/or proline residues.
1. Dose amount 100901 The amounts of the L-asparaginase formulation of the present disclosure that are to be delivered will depend on many factors, for example, the IC50, EC50, the biological half-life of the compound, the age, size, weight, and physical condition of the patient, and the disease or disorder to be treated. The importance of these and other factors to be considered are well known to those of ordinary skill in the art. Generally, the amount of the L-asparaginase formulation of the present disclosure will be administered at a range from about 1 milligram per square meter of the surface area of the patient's body (mg/m2) to 1,000/m2, with a dosage range of about 10 mg/m2 to about 100 mg/m2 to treat disease, including but not limited to ALL or LBL. Of course, other dosages and/or treatment regimens may be employed, as determined by the attending physician.
100911 In some embodiments, the method comprises administering the recombinant L- asparaginase of the present disclosure at an amount from about 10 mg/m2 and about 100 mg/m2. In some embodiments, the method comprises administering the L-asparaginase formulation of the present disclosure at an amount from 10 mg/m2 and 100 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered in an amount of about 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 95, or 95 mg/m2 or an equivalent amount thereof (for example on a protein content basis).
In a more specific embodiment, the L-asparaginase formulation of the present disclosure is administered at an amount selected from the group consisting of about 10, 20, 30, 40, 50, 60, 70, 80, 90, and about 100 mg/m2. In another specific embodiment, the L-asparaginase formulation of the present disclosure is administered at a dose more than or equal to about 1, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 95, 100, 200, or 300 mg/m2. In another specific embodiment, the L-asparaginase formulation of the present disclosure is administered at a dose less than or equal to about 300, 200 100, 95, 80, 75, 70, 65, 60, 55, 50, 45,40, 35, 30, 25, 20, 15, 10, 5, or 1 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 12 mg/m2 and about 90 mg/m2. In another exemplary embodiment, the recombinant L-asparaginase of the present disclosure is administered in an amount between about 20 mg/m2 and about 80 mg/m2. In another exemplary embodiment, the 1-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the 1..,-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 37.5 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 37.5 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 25 mg/m2 and about 37.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m2 and about 100 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 25 mg/m2 and about 80 mg/m2.
100921 in some embodiments, the method comprises administering the recombinant L- asparaginase of the present disclosure at an amount from about 25 mg/m2 and about 50 mg/m2. In some embodiments, the method comprises administering the I,-asparaginase formulation of the present disclosure at an amount from 25 mg/m2 and 50 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered in an amount of about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/m2 or an equivalent amount thereof (for example on a protein content basis).

[0093] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 20 mg/m2 and about 30 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 22.5 mg/m2 and about 28.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 23 mg/m2 and about 27 mg,/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 24 mg/m2 and about 26 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 24.5 mg/m2 and about 25.5 mg/m2.
[0094] In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 60 mg/m2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 42.5 mg/rre and about 58.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 43 mg/m2 and about 57 mg/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 44 mg/m2 and about 56 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 46 mg/m2 and about 54 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 47.5 mg/m2 and about 52.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 48 mg/m2 and about 52 mg/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 49 mg/m2 and about 51 mg/m2. In an exemplary embodiment, the 1,-asparaginase Formulation of the present disclosure is administered in an amount between about 49.5 mg/m2 and about 50.5 mg/m2.
100951 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 45 mg/m2 and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m2 and about 55 mg/m2.
100961 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 75 mg/m2. In an. exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the recombinant L- asparaginase of the present disclosure is administered in an amount between about 55 mg/m2 and about 60 mg/m2.
100971 In an. exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 47.5 mg/m2 and about 50 mg/m2.
100981 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m2 and about 35 mg/m2.
100991 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m2 and about 95 mg/m2. In an. exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m2 and about 90 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 85 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaf,inase formulation of the present disclosure is administered in an amount between about 50 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 55 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 60 mg/m2 and about 65 mg/m2.
101001 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m2 and about 60 mg/m2. In an. exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 50 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 42.5 mg/m2 and about 57.5 mg/m2.
101011 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 30 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 35 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 40 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 45 mg/m2and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered in an amount between about 50 mg/m2 and about 55 mg/m2.
101021 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 10 mg/m2and about 50 mg/m2, In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 12.5 mg/m2 and about 47.5 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 15 mg/m2 and about 45 mg/m2. In some embodiments, the L-asparaginase fonrnulation of the present disclosure is administered intramuscularly in an amount of between about 20 mg/m2 and about 42.5 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 22.5 mg/m2 and about 40 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 24 mg/m2 and about 39 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 27 mg/m2 and about 37.5 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of between about 30 mg/m2 and about 45 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of about 25 mg/m2. In some embodiments, the recombinant L- asparaginase of the present disclosure is administered intramuscularly in an amount of 25 mg/m2.In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 25 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 37.5 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 37.5 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 25 mg/m2 and about 37.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 30 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 35 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 mg/m2 and about 60 mg/m2 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 50 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 m6/, m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 50 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 55 mg/m2 and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 40 mg/m2 and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 45 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 47.5 mg/m2and about 50 mg/m2. In an exemplary embodiment, the I.,-asparaginase formulation of the present disclosure is administered intramuscularly in an amount between about 30 mg/m2 and about 35 mg/m2.
101031 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 10 mg/m2 and about 95 mg/pi'. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 20 mg/m2 and about 60 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 22.5 mg/m2 and about 57.5 mg/m2.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 25 mg/m2 and about 55 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 27.5 mg/m2 and about 47.5 mg/m2.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 30 mg/m2 and about 45 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 32.5 mg/m2 and about 42.5 mg/m2.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 21.5 mg/m2 and about 38.5 mg/m2.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of between about 36 mg/m2 and about 45 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of about 37.5 mWm2.1n some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 37.5 mg/m2. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 50 mg/m2.
101041 In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m2and about 37.5 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m2 and about 100 mg,/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 25mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m2 and about 35 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m2 and about 95 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m2 and about 90 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m2 and about 85 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 45 mg/m2 and about 80 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 50 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 55 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 60 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m2and about 60 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m2 and about 55 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m2 and about 50 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 42.5 mg/m2and about 57.5 mg,/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 30 mg/m2 and about 75 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 35 mg/m2 and about 70 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 40 mg/m2 and about 65 mg/m2. In an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 45 mg/m2 and about 60 mg/m2. an exemplary embodiment, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount between about 50 mg/m2 and about 55 mg/m2.
101051 In another embodiment, the method comprises administering a recombinant L- asparaginase of the present disclosure that elicits a lower immunogenic response in a patient compared to a non-recombinant L-asparaginase.
2. Dose frequency 101061 In a specific embodiment, treatment will be administered at a dose ranging from about 1 mg/m2 to about 1000 mg/m2, typically about 10 mg/m2to about 100 mg/m2, at a schedule ranging from about three a week to about once a month, typically once per week or once every other week, as a single agent (e.g., monotherapy) or as part of a combination of chemotherapy drugs, including, but not limited to glucocorticoids, corticosteroids, anticancer compounds or other agents, including, but not limited to methotrexate, dexamethasone, prednisone, prednisolone, vincristine, cyclophosphamide, and anthracycline.
101071 The L-asparaginase formulation of the present disclosure can be administered before, after, or simultaneously with other compounds as part of a multi-agent chemotherapy regimen. In a particular embodiment, the L-asparaginase formulation of the present disclosure comprises a protein recombinantly produced in Pseudomonas fluorescens, and more specifically, the L-asparaginase formulation comprises a sequence according to SEQ ID NO: l. In some embodiments, the L-asparaginase formulation of the present disclosure is administered at a dose that depletes L-asparagine to undetectable levels using methods and apparatus known in the art for a period of about 3 days to about 10 days (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 days) for a single dose.
101081 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week. [02471 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week for at least one to three weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for about one to three weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the week for about one to three weeks.
[01091 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week for about two weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for about two weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week for about two weeks.
[01101 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week for two weeks. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days for two weeks.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered on Monday, Wednesday, and Friday of the same week for two weeks.
[01111 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week, continuing until the patient no longer has a disease that is treatable by depletion of asparagine. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, continuing until the patient no longer has a disease that is treatable by depletion of asparagine. 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 no longer has a disease that is treatable by depletion of asparagine.
101121 In some embodiments, the L-asparaginase formulation of the present disclosure is administered three times a week, continuing until the patient decides to end or postpone treatment. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every other day over a period of 5 consecutive days followed by a rest period of 2 consecutive days, continuing until the patient decides to end 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 end or postpone treatment.
101131 In some embodiments, the I,-asparaginase formulation of the present disclosure is administered about every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered every 40 to 58 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 42 to 56 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 44 to 52 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 46 to 50 hours.
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 to 80 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 66 to 78 hours.
In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 68 to 76 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered about every 70 to 74 hours.
101141 In some embodiments, L-asparaginase formulation of the present disclosure is administered as a second line therapy with patients who are hypersensitive to an E con-derived I.,-asparaginase, and/or may have had a previous hypersensitivity to an Erwinia chrysanthemi- derived L-asparaginase.

101151 In some embodiments, the L-asparaginase formulation is administered to the human subject as a substitute for a dose of a long-acting E. con-derived aspar4nase. In some embodiments, six doses of the L-asparaginase formulation are administered to the human subject as a substitute for one dose of the long-acting E. coil-derived asparaginase. In some embodiments, seven doses of the L-asparaginase formulation are administered to the human subject as a substitute for one dose of the long-acting E. con-derived asparaginase. In some embodiments, the long- acting E. coil-derived asparaginase is pegaspargase. In a further embodiment, the six separate doses may occur over a period of about two weeks. In another further embodiment, the seven separate doses may occur over a period of about two weeks.
101161 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle comprises a first dose, a second dose, and a third dose, wherein the cycle is optionally repeatable, and wherein the first dose, second dose, and third dose are administered about 48-72 hours apart.
101171 In some embodiments, dose amounts may vary within the cycle.
101181 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 25 mg/m2, the second dose of the cycle is 25 mg/m2 and the third dose of the cycle is 50 mg/m2, followed by the rest period of two consecutive days.
101191 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 25 mg/m2, the second dose of the cycle is 25 mg/m2 and the third dose of the cycle is 37.5 mg/m2, followed by the rest period of two consecutive days.
101201 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m2, the second dose of the cycle is 37.5 mg/m2 and the third dose of the cycle is 37.5 mg/m2, followed by the rest period of two consecutive days.
101211 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m2, the second dose of the cycle is 25 mg/m2 and the third dose of the cycle is 37.5 in mg/m2, followed by the rest period of two consecutive days.
101221 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, wherein the cycle is optionally repeatable, and wherein the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, wherein the first dose of the cycle is 37.5 mg/m2, the second dose of the cycle is 25 mg/m2 and the third dose of the cycle is 25 mg/m2, followed by the rest period of two consecutive days.
in some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is given on a Wednesday, and the third dose of the cycle is given on a Friday.
101231 In some embodiments, a dose regimen for the L-asparaginase formulation comprises a cycle, where the cycle is optionally repeatable, and where the cycle comprises administration of the L-asparaginase formulation every other day over a period of five consecutive days followed by a rest period of two consecutive days, where the first dose of the cycle is 25 mg/m2, the second dose of the cycle is 25 mg/m2 and the third dose of the cycle is 50 mg/m2, followed by the rest period of two consecutive days. in some embodiments, the first dose of the cycle is administered on a Monday, the second dose of the cycle is given on a Wednesday, and the third dose of the cycle is given on a Friday.
101241 in some embodiments, the present disclosure provides a method for depleting asparaginase in a human subject to treat Acute Lymphoblastic Leukemia (ALL) or Lymphoblastic Lymphoma (LBL), the method comprising: on Mondays, Wednesdays, and Fridays, administering intramuscularly about 25 mg/m2 of L-asparaginase to the human subject, such that the human subject receives a total of seven doses of L-asparaginase over a two week period.

101251 In some embodiments, the present disclosure provides a method for depleting asparaginase in a human subject to treat Acute Lymphoblastic Leukemia (ALL) or Lyrnphoblastic Lymphoma (LBL), the method comprising: (a) on Mondays and Wednesdays, administering intramuscularly about 25 mg/m2 of L-asparaginase to the human subject, and (b) on Fridays, administering intramuscularly about 50 mg/m2 of L-asparaginase to the human subject; such that the human subject receives a total of six doses of L-asparaginase over a two week period.
101261 The dose regimen may encompass any number of cycles for any number of weeks or until any endpoint that is specified herein.
101271 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of (i) 25 mg/m2 on Monday, (ii) 25 mg/m2 on Wednesday, and (iii) 50 mg/m2 on Friday.
101281 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 25 rng/rn 2 every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 37.5 mg/m2every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intravenously in an amount of 50 mg/m2 every 48 hours.
101291 In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 25 mg/m2 every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 37.5 mg/m2 every 48 hours. In some embodiments, the L-asparaginase formulation of the present disclosure is administered intramuscularly in an amount of 50 mg/m2 every 48 hours.
101301 In some embodiments, in conjunction with embodiments above or below, the L-asparaginase formulation of the present disclosure is administered (i) intravenously in an amount of 25 mg/m2 on Monday, (ii) intravenously in an amount of 25 mg/m2 on Wednesday, and (iii) intramuscularly in an amount of 50 mg/m2 on Friday.
101311 For example, a dosing regimen may comprise administration of six doses (e.g., a first dose, a second dose, a third dose, a fourth dose, a fifth dose, and a sixth dose) of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously or intramuscularly in an amount of 25 mg/m2 or 37.5 mg/m2 on Monday, (ii) a second dose is administered intravenously or intramuscularly in an amount of 25 mg/m2 37.5 mg/m2 on Wednesday, and (iii) a third dose is administered intravenously or intramuscularly in an amount of 25 mg/m2 37.5 mg/m2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on Friday.

In some embodiments, a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 25 trig/m2 on Monday, (ii) a second dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 25 mg/m2 on Friday;
and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on Friday.

In some embodiments, a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intramuscularly in an amount of 25 mg/m2 on Monday, (ii) a second dose is administered intramuscularly in an amount of 25 mg/m2 on Wednesday, and (iii ) a third dose is administered intramuscularly in an amount of 25 mg/m2 on Friday;
and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on Friday.

In some embodiments, a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 37.5 mg/m2 on Monday, (ii) a second dose is administered intravenously in an amount of 37.5 mg/m2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 37.5 mg/m2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on Friday.
101351 In some embodiments, a dosing regimen comprises administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intramuscularly in an amount of 37.5 mg/m2 on Monday, (ii) a second dose is administered intramuscularly in an amount of 37.5 mg/m2 on Wednesday, and (iii) a third dose is administered intramuscularly in an amount of 37.5 mg/m2 on Friday; and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on :Friday.
101361 In some embodiments, a dosing regimen comprise administration of six doses of the L-asparaginase formulation over a period of two weeks, wherein during week one, (i) a first dose is administered intravenously in an amount of 25 mg/m2 on Monday, (ii) a second dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (iii) a third dose is administered intravenously in an amount of 50 mg/m2 on Friday;
and during week two, (iv) a fourth dose is administered intravenously in an amount of 25 mg/m2 on Monday, (v) a fifth dose is administered intravenously in an amount of 25 mg/m2 on Wednesday, and (vi) a sixth dose is administered intramuscularly in an amount of 50 mg/m2 on Friday.
Kits 01371 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 that is treatable by asparagine depletion in a subject in need thereof.
101381 In some embodiments, the kit comprises a formulation, comprising: (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO:
1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than 2% high-molecular-weight (HMW) species after storage at 40 "C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species and less than 2% high-molecular-weight (HMW) species after storage at 40 C for two months. In some embodiments, the L-asparaginase formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 "C for one week.

In some embodiments, the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ II) NO:
1; (ii) one or more disaccharides, wherein the one or more di saccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week.
10110]
In some embodiments, the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID NO:
1, and wherein the L-asparaginase is present at a concentration of around 20 mg/mL; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM;
and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v), wherein the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø

In some embodiments, the kit comprises a formulation, comprising (i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO:
1, and wherein the L-asparaginase is present at a concentration of around 20 mg/m1.,; (ii) trehalose, wherein the trehalose is present at a concentration of about 170 mM; (iii) sodium phosphate, wherein the sodium phosphate is present at a concentration of about 20 mM; (iv) sodium chloride, wherein the sodium chloride is present at a concentration of about 50 mM;
and (v) polysorbate 80, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v), wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C for one week. In some embodiments, the formulation has a pH of between about 4.0 and about 8.5. In some embodiments, the formulation has a pH of about 7Ø

In some embodiments of the foregoing, the kits comprise instructions for treating cancer. In some embodiments, the kits comprise instructions for treating acute lymphoblastic leukemia (ALL). In some embodiments, the ALL is relapsed. In some embodiments, the kits comprise instructions for treating lymphoblastic lymphoma (LBW. In some embodiments, the LBL is relapsed.

EXAMPLES
Nessler Activity [01431 Asparaginase activity was measured by Nessler assay, in which the measurement of asparaginase activity is based on an endpoint limit assay in which the enzyme is incubated at +37 "C under saturating L-asparagine concentration for 15 minutes.
The reaction is stopped by addition of Nessler's reagent and the amount of ammonia produced during the reaction is assessed colorimetrically (at 450 nm) using a calibration curve constructed from known quantities of ammonium sulfate.
Size Exclusion Chromatography (SEC) 101441 The column was conditioned with mobile phase (50 mM
phosphate, 200 mM NaC1, pH 7.0) at 1 rnUmin for 1 hour before injections. BioRad Gel Filtration Standards were diluted 10-fold in blue dextran for 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 mobile phase blanks to clear the column.
101451 Note that, in the various Tables in the Examples below, the entry "ND"
indicates that a particular data point was not determined.
Example 1: Baseline Buffer/Excipient Screen [01461 The effects of pH and buffer type from six buffer/pH
combinations in the range of 4.5 ¨ 8.0 were assessed on stability of L-asparaginase. An additional set of formulations with 6 different excipients in either 20 mM histidine buffer at pH 7.0 or 20 mM
sodium phosphate at pH 8.0 were prepared as an initial excipient screen (Table 1). L-asparaginase (1.0 mL of Lot RM-LAP-P03/P05/P06 Pool of Bulks at 20.9 mg/mL) was buffer exchanged using centrifugal ultratiltration devices (Amicon Ultra-4 10k MWCO) with 100 mL of the corresponding formulation buffer. Following 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 a one week stability study was performed with the formulated RC at 37 C and 5 C. The formulations were analyzed in parallel at the end of the one week storage.

'fable 1 Sig(ORM(MMRPWRMAM(RWMINMPARROMMTM:NME01.Ma*Ma4Aiiitut:6iilk:MeM
MmummommommomommummummummEmmrõ...mgook_.,,,,mm..,,, õmomminoNM
mi ::x..*irmorrtrotiolf.cdA
1)..igIINA...*.....ik P
...E,222,,EiflgM=Et..k.,i.i(i.1:22g2MMEM2S2E Iiiiii.......10......0=12.ii.
' 45 '. . .
4.6 .. .22 Acetate (20mM) 5,5 5.6 '22 4,5 4.6 Succinate (20n11\4) 5,..5 5.6 5,0 5,1.
2.1 Sodium Citrate

6,0 (20mM', N/A.
6.9 6.0 6Ø

Histidine (20mr411)

7.0 7.1 6.5 i::.i."::$

Phosphate (20m1A) 7.5 7,419 6.0 7...9 .18 Tris (20mtyl) 6õ.5 8,5 3% (i.v/v) Prone 7,1.

150mt,õ1 NaCi 6,9 150m lvi ArO:nino 6.9 2OrriM Histidine 7.0 250mM Trehase 7...1 250mM Sorbitn 7.1 1---., z ..
250mM Sros 5.96 150mM Lysi no 7õ.97 150-inM Nall 7õ89 150rAl Arginine 7...88 20mM Phosphate 8.0 250mM Tre.h&ose 7,90 250rnM Sorbitol 7.87 2.1 250mM Sucrose 7.85 101471 The purpose of the excipient screen was to evaluate acetate, succinate, citrate, histi dine, phosphate, and tris buffers from pH 4,5 to 8.5.
Additionally, the excipients proline, lysine, sodium chloride, arg,inine, trchalose, sorbitol, and sucrose in histidine buffer at pH 7.0 or phosphate buffer at pH 8.0 were evaluated for the influence of excipients. SEC
and the Nessler activity assay were used to assess the stability of RC in the presence of these excipients in the selected buffers.

101481 The purity of 26 formulations was assessed by SEC-HPLC. Results are summarized in Table 2 and Table 3. Little difference was observed in the SEC
profiles between the 5 C and 37 C conditions after 1 week storage for all formulations.
However, differences were observed between the different formulations. The main peak purities had a range of 66.4 98.3%. The histidine buffer formulation all had purity levels between 66.4%
and 74.0%, while the remaining formulations had purity levels between 90.6%
and 98.3%. In the hi stidine, succinate, citrate, phosphate with lysine, and phosphate with arginine buffers, a LMW species was observed at ¨11.6 minutes ranging from 1.3 ¨ 32.4%. The formulations in acetate, phosphate, and tris buffers showed between 0.2 ¨ 0.6% LMW species.
Formulations in phosphate buffer with sodium chloride, trehalose, sorbitol, and sucrose showed similar levels of LMW species to the formulations without excipients.

Table 2 Average MN
Main Peak age ' HMW
Ma Percentage Percent ! Con Average Average : Buffer a PH (GC) (0.4) ' . Condition 0.5 Percentage Percjyri (%) 97.7 , 1.8 0.6 97.9 4.5 37 1.5 0.6 37 1.7 97.7 5 1.7 0.5 97.8 20mM Acetate 5.5 5 1.7 1.5 96.8 1.5 96.9 1.6 4.5 37 1.3 96.8 2.4 20mM 5 1.8 .37 1.6 96.0 1.4 96.8 Succinate 5 1.8 2.0 96.3 6.5 37 1.7 2.8 95.3 5 1.8 2.9 95.8

8.0 37 1.6 3.1 98.2 5 1.8 3.2 95.2 20m104 Sodium 6.0 2.5 95.7 Citrate 37 1.6 5 1.8 2.6 95.7 7.0 37 1.7 27.0 1.3 71.7 28.1 72.7 8.0 ___ 37 1.1 70.7 8.0 20mM
1.3 5 72.2 26.8 Histidine 7.0 __ 37 1.0 0.3 97.9 1.8 0.2 98.1 6.5 37 1.6 0.4 97.8 0.2 20mM 5 1.8 7.5 37 1.6 98.2 0.3 97.8 Phosphate 5 1.8 0.3 98.2 ..
8.0 __ 37 1.5 0.3 97.9 5 1.8 0.2 98.3 8.5 37 1.5 20mM Tris Table 3 Aiter'age ! Average Main Average il NESOMII. ' ' 1 ConditiPn'.... . HMW Peak .9.P.4tmso Exc.iPien eq.
percentage percentage percentage il (74. (74õ,1. .47,9.......J
1,2 67.9 30.9 3% (wiv) Proline 37 , 1.0 , 66.4 , .. 30.0 5 1.4 69.0 29.6 150mM NaCI 37 1.1 69.7 29.2 5 1.3 66.4 32.4 20mM 150mM Arginine 37 1 1.1 60.9 32.0 1 Histidine.
5 1.2 68.2 30.7 pH 7.0 250mM Trehalose 37 1.0 68.7 30.3 , 5 1.3 71.1 27.6 250mM Sorbitol 37 1.0 74.0 25.0 5 1.2 71.4 27.4 250mM Sucrose _________________________ 37 1.1 71.8 27.2 5 1.7 95.9 2.4 i 150mM Lyaine 37 1.4 96.4 i 2.2 fi 5 1.9 97.8 0.3 150mM NaCI
37 1.5 98.3 0.2 5 ! 1.7 90.6 7.7 :
20mM 150m1V1Arginine !-37 1.3 ! 91.2 , 7.5 Phosphate, 5 1.8 97.9 0.3 pH 8.0 250mM Trehatose 37 1.8 98.0 0.2 5 1 1.9 !
97.8 0.3 i 250mM Sorbitol :
37 1.7 1 96.1 0.1 !
5 1.8 97.9 0.3 250mM Sucrose 37 1.6 98.1 0.3 _ 101491 Based on the highest purity and lowest LMW species percentage, acetate and phosphate buffer were selected for further evaluation. Additionally, trehalose, sorbitol, and sodium chloride were chosen as the excipients to be evaluated in the DOE
to assess their impact on formulation stability under more stringent stress conditions.
Example 2: DOE
101501 A statistical design of experiment (DOE) approach considering one numeric factor (pH) and one categorical factor (excipient) was used to evaluate the effects of pH and excipient for acetate and phosphate buffer in the range of 4.5 5.5 and 7.0 8.0, respectively, on L-asparaginase. In order to accommodate the non-overlapping ranges of the two buffer systems, acetate and phosphate, two independent DOEs using a linear one-factor design were performed for each buffer system in parallel. The experimental design was generated using Stat Ease, Inc. Design-Expert Version 9Ø6.2. Significance of the responses were evaluated at 95% confidence level.

101511 An additional set of off-design formulations evaluated the effect of the three selected excipients using Tris buffer at pH 8.5 and the effect of addition of PS-80 to acetate pH 5.3 and phosphate pH 7.5 DOEs with sorbitol and trehalose. The formulations were exposed to a thermal stress, 40 C for 2 weeks, and a single Freeze/Thaw cycle.
101521 The Nessler activity assay for formulations exposed to a single freeze/thaw cycle or stored for 2 weeks at 40 C had activities that ranged from 600 to p.mol/min.mgE (Table 4 and Table 5). No significant loss of activity of the formulations was observed the thermal or freeze/thaw stress, and it is undetermined if the formulations with increased activity is a result of the formulation buffer or the variability of the Nessler Activity assay. The formulations were analyzed by 11P-HPI.,C. :By RP-1-1PLC, all formulations in both acetate and phosphate buffer were determined to be 100% main peak after a single freeze/thaw stress, and 98% main peak and 2% minor peak after 2 weeks at 40 C.

Table 4 Enzyme Aetwity Enzyme Actnnty Buffer PH Excipient (pmolimin.mgE) (pmatirnin.rnsiE) ___________________________________________________ Freeze/Thaw ___ 4OC 75% RH
7.00 726 331 7.50 250 mM Sorbito 773 1270 8.00 718 748 7.

20 mtsil Phosphate 7.50 250 a-1M Trelialose 744 723 8.00 800 300 7.00 7.50 150 mtvl Neel 873 985 8.00 740 878 4 .50 5.00 250 ran Sornitoi 809 796 5.50 719 732 4,50 20 rntvl Acetate 5.00 250 nitil Trehalose 722 676 5.50 691 579 4.50 826 5.00 150 nAl NaCi 1001 763 5,50 Table 5 =n2yrne=
Enzyme Activity = Activ4y Buffer Exc.ipient Surfactant (prpolfrnin.rtigE) (prnoUmitt mgEi FreezeiThaw 40'C 75'"/6 RH
250 rri1V1 Trehalose 702 835 20 mly1 Iris 8.50 250 mM Sc.)rMol 824 150 mrvi Naa 830 709 20 mM 250 mM Sor13101 744 7.50 Phosphate 250 WO Trehalose 0.02% PS- 673 250 ml\sil Sorbtof 80 20 m1V1 Acetate 5.25 _______________________________________________ -250 rriM Trehalose 921 All formulations and stress conditions were analyzed by SEC-HPLC.
Results are shown in Table 6. After thermal stress, the purity in phosphate buffered formulations ranged from 88.70% to 99.31%, the HNINV peak ranged from 0.66% to I 00%, the I.,MW shoulder ranged from none detected to 10.98%, and the LMW peak was none detected. At pH 7.0, all three excipients show similar SEC profiles after thermal stress, and the main peak percentage was 99% for each excipient.

Table 6 HMW Peak Main LmiAlar""""`""T
' *n.
- LMVV ..
I! !pH Extipient . '. Condition ::::: Peak ( %) Shoulder .
%)...õ
.. .]:]::.................. (%) .........A!!!!....reak (%"
Freeze/Thaw 0.68 99.32 ND ND
7 40'C, 2W 0.99 99.01 ND
ND
.0 Freeze/Thaw 0.71 99.29 ND Nn 40-C, 2W 1.00 99.00 ND
ND
250 mM Freeze/Thaw 0.66 99.34 ND ND
7,5 Sorbitol 40'e, 2W 0.89 09.11 ND
ND
Freeze/Thaw 0.71 99.29 ND Nn 8.0 40'C, 2W 0.98 90.33 8 68 ND
Freeze/Thaw 0.66 99.34 ND ND
40'C, 2W 0.94 90.12 8.94 ND
Freeze/Thaw 0.66 99.34 ND ND
40'C, 2W 0.76 99.24 ND
ND
7,0 Freeze/Thaw 0.66 98.05 ND 1.28 40C, 2W 0.75 99.25 ND
ND
7..m 250 mikil Freeze/Thaw 0.68 98.47 ND 0.85 Trehalose 40 C, 2W 0.80 92.44 6.76 ND
Freeze/Thaw 0.68 99.32 ND ND
40'0, 2W 0.82 91.09 8.09 ND
B.o Freeze/Thaw 068 98 44 ND 088 40-0, 2W 0.81 91.36 7.83 ND
Freeze/Thaw 0.68 99.32 ND ND
40 C, 2W 0.69 99.31 ND
ND
7.0 Freeze/Thaw 0.69 (:19,-;,'1 ND ND
40 C, 2W 0.73 99.27 ND
ND
150 mM Freeze/Thaw 0.68 99.32 ND ND
7.5 Nael 40 C, 2W 0.73 92.00 7.27 ND
Freeze/Thaw 0.68 99.32 ND ND
40 C, 2W 0.72 88.29 10.98 ND
5.0 Freeze/Thaw 0.66 99.34 ND ND
40 C, 2w 0.66 88.70 10.64 ND
1011541 The decrease in main peak purity and increase in UAW
shoulder percentage were fit to a linear model dependent on pH only with the maximum main peak purity observed at pH 7.0 (Table 7). The off-design formulations at pH 8.5 also show formation of significant quantities of LIMAN shoulder after thermal stress (Table 8). The formation of HMW species was found to be dependent on excipient, but not pH, with sorbitol having the overall largest percentage of HMW species.

Table 7 otikeVi of"".. tt'f'" Mean "P"":
Squares Square Value POI) F,J!
Model 253.83 1 253.83 83.23 <0.0001 A-pH 25.3,83 1 253,83 83,28 <0.0001 Residual 39,62 13 3.05 Table 8 H PAW- --Main LLIW
Buffer pH Excipient Surfactant Condition peak Peak Shoulder Peak 250 mM
Freeze/Thaw 0.66 99.34 ND ND
Trelialose 40C, 2W 0.71 90.41 8.88 20 mM 250 mM Freeze/Thaw 0.67 99.33 ND ND
8.5 Ths Sorbttol 40"C, 2W 0.79 90.18 9.04 ND
150 mM
FreezeiThaw 0.68 99 32 ND ND
Naa 40'/C, 2W 0.64 86.75 12.61 ND
250 mM
ND
FreezeiTha,,,' 0.91 99.09 ND
Sorbitol mM. 7.5 __ Phosphate 250 !TIM ND
ND
Freeze.iThaw 0.89 99.11 Trehalose 0.0'.)% Ps_ 250 mM
Freeze/Thaw 0.09 97.62 1.69 20 mM Sorbitol 5.3 ____ Acetate 250 mM ND
Trehalose FreezeiThaw 0.6 97.77 1.63 101551 Under freeze/thaw stress for the sodium phosphate formulations, the SEC-IIPLC main peak. purity range from 98.05% to 99,34"A, the IINPAT peak ranged from 0.66%
to 0.71%, and the LMW peak ranged from none detected to 1.28%. No trends were found for SEC under freeze/thaw stress, but the LA/PvI,/ peak was only observed in trehalose formulations.
[0156] Under freeze/thaw stress for the sodium acetate formulations, the SEC-1-111)1_,C main peak purity range from 97.69% to 98.07%, the MAW peak ranged from 0.54%
to 0.69%, and the UMW peak ranged from 1,34% to 1,62%. Results are show in Table 9. No trends were found for SEC under freeze/thaw stress. Under thermal stress, the acetate formulations purity ranged from 97.65% to 98.02%, the IIMINV peak ranged from 0.46% to 0.68%, and the ILMW peak ranged from 1.52% to 1.82%. The small differences in HMW

percentage were found to be dependent on pH and excipient with higher 1-1MW
species at pH
5.5 and more for sodium chloride over trehalose or sorbitol.
Table 9 LMW
]g =-= =":. HMW Peak 7 Main: -::::.'= Lmw PH Excipient ':: Con:dition Shoulder ''*i: T:...................... ........ T.. ....
.............. .................... ... = ( ==== =-= .......
1 Freeze/Thaw 0.61 98.05 1 ND 1.34 40' n , 2W 0.49 97.84 ND
1.67 4.
Freeze/Thaw 0.58 98.07 ND 1.35 40 C, 2W 0.46 97.94 ND
1,60 250 m M Freeze/Thaw 0.54 98.06 ND 1,41 5.0 Sarb!toi 40C, 2W 0.49 97.85 ND 166 Freeze/Thaw 0_58 98_00 ND 1 44 40 C, 2W 0.57 97.83 ND
1.59 5.5 Freeze/Thaw 0:55 98.01 ND 1.44 40 C, 2W 0.56 97.91 ND
1.53 Freeze/Thaw 0.59 98.06 ND 1.35 40 C, 2W 0_47 97_85 ND
1.68 4.5 Freeze/Thaw 0.58 98.05 ND 1.37 40 C. 2W 0.46 98.02 ND
1.52 250 m M Freeze/Thaw 0.55 98.02 ND 1.43 5.0 Trehalose 40 C, 2W 0.49 97.79 ND
1.71 Freeze/Thaw 0.56 98.00 NE) 1.44 5 40 C, 2W 0.54 97.82 ND
1.64 .5 Freeze/Thaw 055 98.03 ND 1.42 40 C, 2W 0.51 97.86 ND
1.63 Freeze/Thaw 0.63 97.94 ND 1.42 4 40 C. -,W 0.54 97:88 ND
1.58 .5 Freeze/Thaw 0.63 9790 ND 1.46 40 C. 2W 0.53 9765 ND
1.82 5.0 150 mM Freeze/Thaw 0.67 97.90 ND 1.43 NaC1 40 C. 2W 0.61 97.76 ND 1.63 Freeze/Thaw 0.68 97.83 ND 1.49 5 40 C, 2W 0.68 97.74 ND
1.58 5.
Freeze/Thaw 0:69 97.69 ND 1.62 40 C, 2W 0:67 97.72 ND
1,60 (01571 All formulations and stress conditions were analyzed by IF X-1-1PLC. After thermal stress, the phosphate buffer formulations showed a main peak purity range from 52.9% to 63.1%, the acidic species from 9.3% to 18.8%, and basic species from 25.2% to 30.1%. The amount of acidic species increased, while the main peak decreased with increasing pH. The main peak percentage was also dependent on the excipient type. Results are shown in Table 10 and Table 11.

Table 10 Peak HIVIVII = Main ":' '7u LMIN i i!
pH Ekcipient . Condition Shoffider 7 .1:.:.:.:.:.:.:.:.:.a.:.:.:.:.:.:.:..:.:.:.:.:.:.:.:.:.:.:.:..:.:.:.::.A.:.:.
Freeze/Thaw 0.61 96,05 ND
1.34 40C, 2W 0.49 97,84 ND
1.67 4,5 Freeze/Thaw 0.58 98,07 ND
1.35 40C, 2W 0,46 97.94 ND
1.60 250 mM Freeze/Thaw 054 98.06 ND
1.41 5.0 Sarbitol 40C, 2W 049 97.85 ND
1.66 Freeze/Thaw 0.56 98.00 ND 1 40C, 2%Ac 0.57 97,83 ND
1,59 c c Freeze/Thaw 0.55 98,01 ND 1 40"C, 2W 0.56 97.91 ND
1.53 Freeze/Thaw 0.59 98,06 ND
1.35 4 40C, 23,N,' 047 97.85 ND
1.68 ,5 Freeze/Thaw 0.58 98.05 ND
1.37 40C, 2W 0.46 98.02 ND 152 250 mM Freeze/Thaw 0.55 98.02 ND
1.43 5,0 Trehalose 40'C, 2W 0.49 97,79 ND
1,71 Freeze/Thaw 0.56 98,00 ND
1.44 40 C, 2W 0.54 97,82 ND
1.04 5.5 Freeze/Thaw 0.55 98.03 ND
1.42 40C, 2W 0.51 9785 ND 1 Freeze/Thaw 063 97.94 ND
1.42 4 40C, 2W 054 97.88 ND
1.58 .5 Freeze/Thaw 0.63 97.90 ND
1.46 40-C, 2W 0.53 97 65 ND 1 150 mN/I Freeze/Thaw 0.67 97.90 ND 1 4 ':', 5,0 NaCl 40'C, TA' 0.61 97,76 ND 1.63 Freeze/Thaw 0.68 97,83 ND
1.49 4042, 2µ,PV 0.68 97.74 ND
1.58 5.5 Freeze/Thaw 0.69 97.69 ND
1.62 40C, 2VV 0.67 97.72 ND
1.60 Table H
.:.: = Acidic: -::-:
N:lan ,:.
:.:.:. .... ==! 'Buff* : :pH Excipient Species Main Peak Basic Species (%) Peek RT"."
CIO Cl:..0 (P-tin) 13.5 57.5 29.0 18.6 7.0 13.3 57.2 29.5 18.6 250 m M
7.5 16.3 56.7 29.0 18.6 Sorbitol 18.6 53.2 28.2 18.6 18.8 52.9 28.3 18.6 10.6 59.3 30.1 18.7 7.0 10.3 59.9 29.9 18,7 20 mM 250 mM
7 __________________________ 5 12.8 57.8 29.4 Phosphate Trehaose 15.0 55.7 29.3 18.6 8.0 17.7 57.0 25.2 18.6

9.5 63.1 ,,7...:,.
, ..L. t 18.7 9.3 62.1 28.6 18.6 150 mM
7.5 11.9 59_2 28.9 18.7 NaCi 0 13.8 56.6 29.6 18.7 8.' 15.5 56.1 28.4 18.7 6.5 54.6 38.8 18.7 6.2 54.1 39.7 18.7 250 rnM
5.0 6.1 54.0 3..., -6 .,..a 18.7 Sorbitel 6.6 55.2 38.2 18,7 Jr r . J
6.5 5.5.3 - R n ,),...., 18,7 6.4 53.6 40.0 18.7 6.3 54.5 39.2 19.3 250 mM
20 mM Acetate 5.0 Treha]ose ..,-., 6.2 ...),._...-,.. 38.6 19.3 6.2 56.3 37.5 19.3 5.5 5.9 53.3 40.8 19,2 4.5 7.3 51.4 41.3 192 Ti 51.3 41.6 19.2 ___________________________ 150 5.0 RIM 6.2 52.1 41.8 19.2 NaCI
6.3 56.4 37.2 19.2 6.2 54.1 39.7 19.2 101581 At pH 7.0, the main peak percentage was 62A -63 .1%, 59.3-59.9%, and 57.2-57.5% for sodium chloride, trehalose, and sorbitol, respectively. No trend was observed for the amount of basic species under thermal stress. A similar trend for pH
was observed for the main peak and acidic species percentages for these formulations under freeze/thaw stress (Table 12), but little difference was observed between the excipients.

Table 12 =7. Buffer,': pH Excent Species Mean Peak Basic ipi Peak T T
T 7.8 -t- 69.5 T 22.7 r 19.2 7.0 7.5 694 21..1 19.2 250 niM
7.5 orbitot 8.6 68.5 22.9 19.2 S
WA 67.4 22.2 19.2 8.0

10.0 67.2 22,8 19.2 7.5 69.1 23.4 19.2 7.0 7,3 69.5 23.2 19.2 20 mM 250 mM
7.5 9,2 '64.1 26.7 19.3 Phosphate Trehalose 9.6 65.3 -?..z,.1 19.3 8.0 10.9 67,4 21.7 6.9 68,6 24 ,, .,,.,, 19.3 7.0 7.1 68.6 24.3 19.3 150 mM
7.5 8.6 66.2 24,5 19.2 Naa 8.7 66.8 24.5 19.2 8.0 10.1 66.4 23.5 19.3 5,5 67.7 26.8 19.2 4.5 5.5 67.7 26.7 250 rhiM
g.0 5.7 68.8 95.7 lg.?
Sorbitot 5.8 59.0 25.3 19.2 5.5 5.8 604 24.8 19.2 5 5.6 69.7 24.7 19.2 4.
5.6 69.7 24.7 19.2 20 rnM Acetate 5.0 250 mM 5.5 70.2 24.3 19.2 Trehalose 5.5 70.4 24.1 19.2 5.5 5.7 69.8 24.6 19.2 5.4 70.4 24.1 19.9 4.5 5.5 70.4 24.1 19.9 hAll 5.0 150 5.5 70,5 24.0 19.9 NaCi 5.6 70.6 23.9 19,2 g 5 5.6 70.4 24.0 19.2 After thermal stress, the acetate buffer formulations purity ranged from 51.3% to 56.4%, the acidic species from 5.9% to 7.3%, and basic species from 37.2% to 41.8% (Table 11 above). The main peak decreased with decreasing pH, and the main peak percentage was also dependent on the excipient type. At pH 5.0, the main peak percentage was 54.0%, 55.2%, and 52.1% for sorbitol, trehalose, and sodium chloride respectively. No trend was observed for the amount of basic species observed under thermal stress. Under freeze/thaw stress in sodium acetate buffers, a similar trend was observed with p1-1 and excipient relative to the thermal stress, although the difference between formulations are relatively small (Table 12 above). The ll-X-HPLC results for the off-design formulations are found in Table 13 and Table 14.

Table 13 x: ====== ==== Acid .:'=-= Main Basic Main '''' i p Buffer pH. Excipient Condition Species Peak (%) Species Peak .. ..... (N) (RT) i 25o mki Trehalose 13.2 57.8 29.0 1.:).2 250 miA
20 mfOTris P, . 5 40"C, 2W 14.2 57.5 28.3 19.2 Sorbitcl 250 mM
13.4 61.6 24.9 13.6 NeCl Table 14 Acid .. Mean .. Basic Buffer pH Excipient Surfactant Species Peak Species Peak rilcl(RT,):::::
250 mM
7,5 69.5 22.8 19.2 Trehalose 250 mM
20 mM -iris 8.5 ---- 7.0 70.1 22.9 19.2 Sorbitol 250 mr,11 7_8 697 22.5 19.4 NaCI
250 mM
9_2 68.1 22.7 19.4 20 mM Sorbitol Phosphate 7. 250 8_6 68.8 22.6 19.4 Trehalose 0.02% PS-80 250 mM
6_2 69.6 24.2 19,5 Sorbaol 20 mM Acetate 5.25 250 6_2 69."-t 24,5 19.5 Treha lose All formulations were colorless and clear after vialing After freeze/thaw stress all formulations were colorless and slightly opalescent (Table 15, Table 16, and Table 17). Under thermal stress for the sodium phosphate formulations, product related precipitate was observed at pH 8.0 for sorbitol, p1-I 7.5 and 8,0 for trehalose, and in all sodium chloride formulations. For the sodium acetate formulations, particulate was observed in all formulations except 250 mM sorbitol at pH 4.5 and 250 mM trehalose at pH 5,5.

Table 15 .................,......... , .:.4.ft-..'s,.?k-==:.pr.6.4,..:..Ø***11**=*.:*.i.:',...:..'..'..:..:..:.:..:..:..:..:..:..:..:
..'..:..:.:.'.'.*4;
::.......z:r.::::::: :::v15 ::.toit,....:.,...f.s:::] ::::::::::::::::::::::::-:::::::::::.:.:.:.:.:.:.:.:.:.:.:.:.:,v1, .4.-.-7.kt:,,I,A:imit.!!tf:.:.:.:.:::.:.:-:.:.:.:.-.......-........................-........................,..............,.................om..:w4...
k....m...:AKI..-........-...............,...............'...::-, .:.:.11f71.??Fxu.3.. ....wiEn.:?:::::::,:::: ... . . :::::::.::::::::::::]...
... . . . .........: .:......:.:.:.:.::.::.:.:.:.:.. = .
:.:.:.:.:.:.:::::=:::::::=:::::::::::::::::::;::=:::::::::::::y...:::::::=:::::
::::::::::::::-.".::::::::.:: .. . = . ..-:..*::',=:=:=.*::,.*:::::=:: :.: :::
=:=====:=::=:=:=:**:=:=:=:=:=:=:=:':====:.:.:.:.:.:,, .6E0.0i.1tit:EA aii0:0011Ø1Wi .iEi...Ø4)0.ti:i:g i::i:i:i0ii4iitiViMMiiPiailiitiiiP:
es Slightly No Visible Slightly No Visible Colo C'o lorle ss 7.0 Opalescent Particulates Opalescent Particulates Slightly No Visible Slightly No Visible Colorless Colorl,,ss Opalescent Particulates -''' Opalescent Particulates 250 olorless rnIVI Slightly No Visib Colorss le ,S.:lightly No Visible 7.5 ' l=
Sorbitol C = Opalescent Particulates ' -''' Opalescent Partictilates Colorless Slightly No Visible Slightly No Visible Colorless , 8.0 Opalescent Particulates C."Ipalescent Partic:riates Slightly No Visible Slightly Colorless '- Colorless - -YES
Opalescent Particulates Opalescent Colorless Slightly No Visib Colorless - =le Slightly No Visible 7 Opalescent Particulates Opalescent Particulates .0 Slightly No Visible _ Slightly No Visible Colorless=Colorlces -Opalescent Particulates ' - Opalescent Particulates 250 mi\.,1 Slightly No Visible Slightly 7.5 Colorless Colorless YES
Trehalose Opalescent Particulates Opalescent Slightly No Visible Slightly Colorless Colorless \ e- ES
Opalescent Particulates CValescent 8.0 Slightly No Visible Slightly Color:less Colorless =
Opalescent Particulates -Opalescent Slightly No Visible Slightly Colorless Colorless L ' YES
Opalescent Particulates Opalescent 7.0 Slightly No Visible Slightly Colorless Colorless YES
Opalescent Particulates -Opalescent 7.5 150 TIM Colorless Slightly No Visible Colorless Partly YES
NaCi Opaiescent Particulates Cloudy Slightly No 'Visible Partly :Colorless Colorless YES
Opalescent Particulates Cloudy 8.0 Slightly No Visible Partly Colorless \
r"-Opalescent Particu Colorless ES
lates ' Cloudy ., Table 16 õ
viii:i:i:i:ii:i:i:i:i:i:i:i:i:i:i:i:i:i:i:i:ii,i:i:i:i:i:i:i:i:i:i:i:i.iii,iiii iiimiiiiimi,,,,,,,,,iii,iii,i,&.===,,.i.._iiii.i.i...i.i.iiii"liii,,i.i.ii:i:ii iimmiiiimmiiminiimmig.irmiiiiiii,iiiiii,i4.i.ii.i.ii,tH=iipi=i.i..rb,==-it,/=.....Mi,i*ii*MMSM, ....::::: t,....,:.:.:.:14.: :1:tol :_;::;::
k:::.:.:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::r illt'imxr ....pf :ient-**,:::.:::::.....,,,..:::::::,::::::::::.::::.::::,...: . ..-- .
,:n::::::::.::::::::::::::::: . :.:..:.:..:..'..:.:-i.-'..].:.:.::-'5i*-..:.:.:..:.., : := :*.' = :. :f.:.:.:. . .:.:.:. ................. . ::::.:. .
::.:::..::::. . ::::**:*: . :**::,--:::::::::::::::::::::::'::::::::]..:."M:::::i' g::::M::::::::::::::::::::::
Colorless Slightly No Visib 'Colorless le Slightly ' = No Visib/e Opalescent Particulates Opaiescent Particulates 4.5 Colorless Slightly No Visible 'Slightly No Visible colorless Opalescent Particulates - " - Opalescent Particulates 250 rni\i'l Slightly No Visible Slightly 5.0 Colorless Colorl Y
Sorbitol Opalescerit Particu ess ESlates = Opalescent Slightly No Visible Slightly Colorless Opalescent Particulates Colorles- Opales.cent YES
.5 Sliclhtly No Visible Silg htly Colorless - ' Colorless YES
Opalescent Particulates Opalescent Sliglatiy No Visible Slight/y Colorless Col,; YES
Opalescent Particu orles lates " - Opalescent Colorless '' Slightiy No visibie Slightly No Visible Colorless Opalescent Particulates ' - -Opalescent Pat C i.i fates 250 m'1\4 Colorless u Slightly No Vib isle 5.0 oiorless YES
Trehalose Opalesce.rit Particulates Opalescent Slightly No Visible Slightly Visible Colorless Colorless - ' = ' '' ' Opalescent Particulates Opalescent Particulates 5.5 Colorless Slightly No Visib Colorless le Sllg htly No Visible Opalescent Particulates " Opalescent Particulates Slighthy No Visible Colorless Colorless Cloudy Precipitate Opalescent Particulates 4,5 Colorless SlkjiTtlY No Visible Colorless Cloudy Precipitate Opalescent Particulates = ' ¨ -5.0 150 mM Colorless Sligh* No Opalescent Particulates'C Visible . .
olorless Partly Cloudy Precipitate ' "
NaCi Slightly No Visible Slightly Colorless Colorless YES
Opalescent Particulates Opalescent 5.5 Slightly No Visible Siightly Colorless - YES
Opalescent Particu Colcirles' lates - Opalescent Table 17 250 :TIM Sghty VieNe õ_, cs.io$1e PartA's,' No VLsit-Ae.
Css.
Trehaoss. Opalescent Pa:t culates Cloudy Parttcustes 2 Stli toM SAty No Viathle =Parry No Vie 20 msh,,ITtis 9.5 ' .= Co:oriess ' Sorbito; Opalescent Pet t;r_i..tlates Cloudy Rerticaetes 150 rnM coinziess .Vightty No Visib:e.
Party Cocraas YES
N Opalescent Particu.iates ' Cloudy 250 rnlrvi tic,lozlese Si.'ightty Sorbatol Opalescent Pa tt cOates.
20 revi 7,5 Filoshete Si:ichty No Visibte Trehaose 0 02% ps_ ''')c'c' e$'5 Opalescent Paztouiates RE'RMMUMnMEM']']']g 250 ;TIM
, .
20 rnm Sorb.itol opalescent ParticQ 31"AS
5,3 Acetate 2 No Vist'ote Colo:less Trehoose opalescent Park...0 i,.,.es 101611 The analytical data for the sodium phosphate buffered formulations under thermal stress was selected for further analysis due to the degree of difference between the formulations, and the client's historical experience with RC in phosphate buffers. The results from the SEC, IEX, and appearance testing showed a clear trend on p1-1 for formulation stability, with the excipient showing a lesser effect for the formulations at pH 7Ø
101621 The numerical optimization was performed for the sodium phosphate formulations under thermal stress using Stat-Ease, Inc. Design-Expert Version 9Ø6.2 in order to maximize the SEC and IEX main peak percentages and minimize the formation of II,MW and ITNINV species in SEC and acidic and basic species in 'EX. The numerical optimization was performed on the analytical results from the 20 naM. sodium phosphate DOE after 2 weeks of thermal stress. The formulation optimization was performed to find the optimal formulation without any constraints on the pH or excipient. The optimization parameters and results are found in Table 18, Table 19, Table 20, and Table 21. When the presence of particulate in the formulations used as a response and all results were given equal importance, the optimal formulation was calculated to be 20 riiM sodium phosphate, 250 rtiM
trehalose at pH 7.2. If the appearance of particulate in the formulation is not factor as a constraint in the optimization and all results were given the same importance, the optimal formulation was calculated to be 20 riiM sodium phosphate, 150 niM sodium chloride at pH

7,0. Based on the DOE, and off-1)()E formulations, the 20 mIME sodium phosphate, 250 mkt trehalose, pH 7.0 at 20 mg/mL RC was selected for further testing in the surfactant study.
Table 18 _ .
4.0*.i.r;i;;i1;i;i140.00.C;ii;i;i;44.4.***Ci;i;i;.4.400.06i;i1;i;i4i;i;i;i;i;i;
i;i:i;i;i;ili;i;ii;i;i1;i;i13 14004..:.0Ei:ZiMiEii:.iiEA0OtaiEiiltgiiii1).tiEiiEit.00.f.i.*iV.VOIdtit i=iN.O.tdri.CENOVittOtWO
A:pH, is ii-i range i 8 1 1 B:Excipient is in range Sorbitoi NaCi 1 1 SEC (1-IMW) minimize 0.66 1 1 1 ..1 SEC (Main) maximize 68.29 99.31 .1 , 1 3 SEC (LMW) mze 0 10.98 1 1 FEX acidic % minimize 9.2587 18.7732 1 1 .:.., 1EX % Main Peak maximize 52.9391 63.1144 1 1 1EX Basic c.%. minimize 25.2444 30.0589 Table 19 $00i!1!1!ii 1!1!RUO ....'!'!'!!'!'i!RiL!'!4Ri .'!!'FIE11!:]!1RI OM
.. ........ .. 1.0-..ik').!!
,,,,.;,;,;,,,;,,,,;,;,;,;
i;ii=i=iii;i=i=i'i.*i.:i=i=i:i=iiii:iii;:H:i:iii;;;.:i:ii:i:ia:::: . ; . .-..,;i;i A;:;:;:ii4M,. . . . .... idi;::::0.,p1pRof to., PIrtii.!!!..:...Pf.7:-F:PJ.:f.!M&Ilii:z;i:z-,:Ai?::;m;f,;:..-.:',':-t:..-... .....:. .
.........Ltiiw,,,Adieltii,,],,,rImp FrIT.. ''''''''' ......i..:......:::::::.
iiiMMIUMNIUMEMENSENINEHMINEHAUtti! ' ' .#0* ' ;:-.;%:;:;:',1;1;11..111111111111 1 7.0 NC 1 0 700 5916 0.016 9.25 62.55 28.07 0.833 2 7.3 Trehaicse 0.788 96.39 2.788 12.20 58.57 29.32 0.521 Table 20 ii= = = = = = = = = =
l.f.f.f.:1:1:1:1.:1:1:1:1:1:1:1:1:1:1:1:1:::.:1:1:1:1:1:1:1:1:1:1:1:1:1:1:17.:1 :1:[1:1:1:W:f:i::i7:7;;;;;:i::i':i::i::i::i':i::i::i7:7:7:i::i::i::i':i::i:
Lowerj.'..'.':=.=':=.=':=::-:=.='r:=.=':=.=':-:.=':=.=':=.=)=).:....:ii=iiiit.ii:)=.'.'..!!2.i.':1:'=':1:':1:1::-Iti..:iiiii#:.''.''.47.1:'i:'i:'i:':','''':,':,':,':,''.:,''.:,'':,'':,''.:,''.
:,'::::,''.:,''.:,''.:,''.:,''.:,''.:,'!,''.:,''.:,''.:,'::::,'':,'':,7:,'':,'' :,1:il Illiglilililililigiaillililiglililillililliililililililililiddiitglilililililil illlilililillLiiiiiitlilililililiilillililitiiiiitt011ililWitiitiell1Witkitiell Aiiiii664664.
................ ................. ............ .................. ...........
................. .....
A:pH is in r.anaie 7 8 1 1 '=-B:Ex.cipient is in ranae Sorbitot NaCi SEC (H1,,,,Mi) mini:mize 0.66 1 1 1 SEC (Main) maximize 38.29 99.31 1 1 SEC (UAW) minimize n 10.98 1 1 1EX acidic (..% minimize 9.2587 18.7732 1 1 Main Peak maximize 52.9391 63.1144 1 1 LEX Basic % minimize 25.2444 30.0589 1 1 ===
Particulate minimize n 1 1 1 Table 21 apt!: SEC igx :
Numb& pH Excipleat % ¨
t4rA' Lmw ACPMQ Main : : :
Peidi( 1 7.2 "rehalc3e 0.798 97.037 2.145 :1.82 53.7,9 29.51 0.332 0.536 2 7.0 Sorbitol 0.959 99.167 0.016 13.16 57.44 29.31 -0.050 0.467 Example 3: Surfactant Study

11.01.63.1 A surfactant screening study was performed to evaluate the impact of PS-80 concentration on observed opalescence 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 between 0.00% and 0.06% w/v (Table 22).
As an alternative formulation, 20 mM sodium acetate, 250 mM Trehalose at pH 5.5 with 0.00% and 0.04 A w/v PS-80 was included.

Table 22 Time. ,Csoncentratitm g!!!!! Buffer Excipient pH PS -80 V.%) Point /Condition (rngimL..) Fir 21 0,00% 3 FIT 21 0 H AVation Control 21 24 H Agitation 21 H .Agitation21 3 Fri 21 O 0.02%
H Agitation Control 21 24 H Agitation 21 7.0 48 H Agitation 21 TO nn I FR-3 Fir 22 0314%
H A4i1ation Control 22 24 H Aultation 22 2.0 rt-Al 2Ei0 flTh 48:H Agitation nn 22.
Sodium .Trehaiose TO
Phosphate 3 FiT 22 0.08%
0 H Agitation Controt 22 24 H Aditation 22 48 H Agitation 22, 1 Ff1 20 6,5 H Agftation Control 19 24H Actitation 20 49 H Aaitation nn 2d 0.04%

1 'FIT 21 0 H AVation C'ontrot 21 24 H Agitation 21 48 H .Aditation 21 Table 22 ((1ont'd) me ]:] Buffer Excipient ps_w Concentration imgifrnl*
]] rfq PointtCondition 3 Err 20 0.00% 0 H Acitation Control 24 H Agitation 20 mhi 48 H Agitation 20 Acetate S aditj ,5 Trehalose TO 21 I Fri 21 0;04% 0 H Aoitation Control 24 H Agitation 21 48 H Agitation 21 11¨asparaginase (at 22 mg/mIõ 1760 mg) was dialyzed into the corresponding formulation buffers without PS-80 using 10K MWC0 Slide-A-Lyzer dialysis cassettes. Dialysis was performed at room temperature with a minimum formulation buffer to sample volume of 40:1. Four dialysis buffer exchanges were performed. Following dialysis, the material was removed from the cassettes, and the concentration and pH were measured. The material was then split and PS-80 was added to the appropriate concentration using 10% (w/v) PS-80 solution in H20. After mixing, each of the formulations will be aliquoted equally into six 2 mit, vials.
[0165]
One vial from each formulation was held at 5 C for the initial time point testing (8 total) during the freeze-thaw and agitation evaluation. One and three freeze-thaw cycles were performed independently on separate vials of each formulation.
Each freeze-thaw cycle was frozen at -75+10 C for a minimum of 2 hours and thawed at room temperature for a minimum of 2 hours and until completely thawed. Three vials from each formulation were required for the agitation evaluation (24 total). One vial was agitated on a rotator at 60 rpm at ambient temperature for 24 hours, one vial for 48 hours under the same conditions, and one vial was placed next to the rotator for the duration of the study as a control.
[0166]
The surfactant study was performed to assess the impact on PS-80 on the development of opalescence under freeze/thaw or agitation stress for RC
formulations in 20 niM phosphate, 250 niN4 trehalose at pH 6.5, 7.0, and 7.5 with 0.00%, 0.02%, 0.04%, and 0.06% PS-80 (Table 22). Two additional formulations were added to the study as potential buffer alternatives: 20 mM acetate, 250 rnM trehalose at pH 5.5 with 0.00% and 0.04% PS-80. The formulations were then exposed to 1 and 3 freeze/thaw cycles at -75 C
and stressed with agitation for 24, and 48 hours at room temperature. Controls for each formulation were held at 5 C for the freeze/thaw stress and room temperature for 48 hours without agitation.
101671 By appearance testing, all control and stressed samples were colorless and slightly opalescence without product related particulate. There was no observed difference in the degree of opalescence between the control samples and the freeze/thaw and agitation samples or the different levels of PS-80. The protein concentration was also assessed for controls and stressed formulations. The protein concentration was between 19 and 22 mg/mL
(Table 22). No significant changes or trends were observed upon freeze/thaw or agitation stress.
101681 The DLS analysis showed that all samples had a hydrodynamic radius between 4.1 and 4.5 with a percent polydispersity between 10.7% and 19.4%
(Table 23 and Table 24). No significant differences or trends were observed in the DLS
relative to formulation or stress condition.

"fable 23 Cdt Time ' di us = = "
TO 4_2 155 1 FIT 4_3 154 3 FIT 43 15.7 0.00% 48H Agititiljon 4.4 17 5 24H Agitation: 4.3 14.5 48H Af_t4itittlion 4.4 15.5 7.0 TO 4.4 154 4.4 15.5 3 :FIT 4.3 144 /3.02% 48H Agitation 4 I 9.4 Control 24H Agitation: 103 48H Atti itation 4.4 14.5 TO 4.5 15.3 FIT 4.3 122 3 Fri 4.3 153 0.04% H Agitation 4.4 17.2 Control 24H Agitation: 4A 15.4 48H .A..q ita 4. 3 447 7.0 TO 44 15.5 FIT 4.4 14 2 8 FIT 4.4 17.3 0.06% 48H Agitation 4 4 14.9 Can trol 24H AgitaIlixt 4.4 14 0 48H Aioon $5. 17 TO 43 15.1 1 Fri $2 11.0 FiT 4.3 131 5.5 48H A?t,1 qation 4 3. 113.7 Cat trol 24H Agitation 4.3 13 7 4:a H AQ qatiDn: 4.73 .04%
TO 4.2 17.1 I :FIT 43 15.7 3 FIT 43 13.5 7.5 43H AgitatiDil 4_3 14 3 Cott:trot 24H AttiAatiort 4.3 I 2 40tH A51tx 4.3 14 6 Table 24 PS-;80 nrne õ - = : -( Y.$ tsCe.nuiftiorn:: P:e-Pr:ge TO 4.3 14.6 1 FIT 4.4 14,2 3 ?VT 4,3 14.0 i).00'-X 4-8H Agitat 4.1 14 4 Control 24H AgitatkiI 4:1 15.6 48H AgitatiDrE 4.2 15,2 5,5 TO 4,3 16.6 15/1 4.3 15,0 351 4.4 15.5 O. 045,.,, 45HAgt>tor 4.3 16 3 C.o.ntmi 24H Agitatjon 4.4 16 48H AgitathDrE 4.4 16,3 101691 The SEC-HPLC results show that all formulations, regardless of stress, in 20 inM phosphate buffer had similar profiles with the main peak percentage of 99.3% and a 1-1MW peak of 0.7% before and after stress conditions (Table 25). Similarly, the SECHPLC
of the 20 mikil acetate formulations showed 0.7% EIMW peak, between 97.9% to 98,1% main peak, and between 1.3% to 1.4% LMW peak (Table 26).

Table 25 Ps.8.0 co :::: ::,...:: "Conditioa::::: i!:. PC Peak t..,,,). : : . = :.::.
,,.......!:!.!.!.!.!.!-.......mv.... .!.!.!.. :... ::........... M.) ...........1........ ........, ............ (%). ........!, I . ..........1....3.:........
To 0.71 09.29 ND
FfT 0_79 -99.39 ND
:3 FfT 0.70 89.30 ND
0.00% 48H Agaziti-c9 U.89 89.31 ND
24H Ar.3.1:atk:m 0.71 99.39: ND
481-1 At:gab-an 0.71 99.23 ND
TO
TO 0.72 99.28 ND
1 F,7 0.71 89.29: ND
:3 FIT 0.72 99.20 ND
0.02% 48H Au.taiar$
0.71 99.23 ND
:Coritml 24H Agitatn 0.72 89.23 ND
48H Ag03tK,?:!3 0.72 99.20 ND
TO 0:71 99,20 ND
1 F.7 0.71 89.29: ND
:3 HT 0_71 99_23 ND
0.04% 48H Aqitati9 0_71 99.2,3 ND
:Cont:PA
249 Aggaticn 0.70 99.3Ø ND
48H AO'atic:9 0.71 199.29 ND
7.0 TO 0_72 99_28.
ND
1 FT 0.72 89.20 ND
.3917 0.73 99.27 ND
0.06% 48H Acgation 071 99.23 ND
Contr-oi 249 Ag;tatc::! 0.70 99.30 ND
439 Aglatiors 0.72 99.28. ND
TO 0.70 -89 .39 ND
1917 0.88 89.34 ND
35(7 0.71 99.29 ND
0.70 99.99 ND
Control 249 Agitatibi 0.70 9930 ND
43H .4',Qtatkan 0:70 09.3.9 ND
0.04%
TO 0.73 0027 ND
1ST 0_73 09_27 ND
OFT 0.71 80.29: ND
AOsation 0.72 99.28 ND
Cc)ntrd 249 Agtati:c.-,9 0.73 99.27 ND
499 .4ta0,o9 n 72 99.28. ND

Table 26 HAIM Peak "
41* PS-80 r): onditicift RC Peak cm TO 0,63 97.95 1.37 1 7/7 0,68 98.00 1.33 3 FT 0.69 98.01 1.30 0.00% 48H Agitation 0.87 98.01 1.32 Control 24H Agitation 0.66 98,06 I.27 48H Agitation 0.67 98.00 1.34 5.
TO 0.68 98.03 1.29 1 FIT 0..88 97.92 1.40 3 F/T 070 97.94 0.04% 48H Agitation 0,68 98.00 1.32 Control 24H Agitation 0,68 97.99 1.33 48H Agitation 0.69 97 1.34 [0170] Sub-visible particulates were measured by ILIAC for the TO, 1 freeze/thaw cycle, 3 freeze/thaw cycles, agitation control, 24 hour agitation, and 48 hour agitation samples with varying concentrations of PS-80. The results are summarized in Table 27 and Table 28 For all samples the cumulative counts/mL ranged from 2330 ¨ 45270, 390 ¨
35857, 78 ¨ 6430, and 0 ¨ 150 for ?. 2 1.un, 5 1.tro, 10 pin, 25 /Ail, respectively, A few conditions had elevated particulate levels relative to the other conditions of the same formulation, but these samples appear to be outliers rather than representing a trend For example, 20 mM phosphate, 250 mM trehalose, 0.04% PS-80, pH 7.0 solution had >10um particles at the initial timepoint, but only 155 particles in the agitation control Table 27 Tm 1.0t :.: FS-e0 t''.4,M t.ti .::n.,..c.,..,d:tiolt:
... ..õ , Mr.
:.....:::::::1........... i::i:.. :..:................::::::i:i 9 ", -~
= ' TO 154343 265.,0 522 1 FT 45270 21i380 0430 150 3 Fri 8.538 1447 313 8 0.00%
0 H A.citz.ition ContDai 181.95 3540 755 24 H Agitatio-fl. 5145 8135 157 .2 48 H AOation 18402 3172 .578 5557 1027. ..8.8

12 0.02%
0 H Ag tation C Kmi.ln-A 3028 49e 105 24 H .4o:Ita03n: 4350 815 152 =-) 48 H Aqjt.ati,)-8 17732 3103 4'.a9 1.2-:
7.0 35:'F 5730 1e0 252 10 0.04%
0 H AcAtation Conirck 3123 033 155 24 H AVation 9040 2122 457

13 48 H Agrt.abcm 13538 35857 869 62.30 117.2 260 0..05%
0 H A.0,31ion "Thnird 7503 1602 327 e 24 H .A.g.itation 425D .028 1138 48 H Ao:I.anoo, 20525 4120 653 3 F7T 23.30 320 TO t'D
6,5 0 H At-,=tialk:11-$ Ggrlifd 3555 903 156 24 H .AØ3-tic.3-:. 9077 2353. 495 5, 48 H Agi1atio.n 11258 148.3 '190 0..04%
TO 3355:3 6140 1243 1 Fri 7458 1108 18.5 5 7.5 0 H Aq.italion Cc31.:tro1 2840 457 135 24 H Agitatic,n 15616 2566 443 413 H Ag6ation 19282 3733 322 'The sample testing .was interrupted and did not fuqy comOete. However: the measurernent(s) taken represent ectua resuft for sample Table 28 lll :4K..:t l PS-80 OW , = ,T,..!:11 - = t* W *it OWN g W0400 *45 OW

OH
0.00% Agtation 8667 1432 353 23 Control Agitation 11217 2040 353 23 A.gtatior.
5, ':', 7.:1-. 12148 2190 -:.,--,.> 1-,,-, OH
0.04% Agitation 2955 600 110 2 Control Agitation 14297 2437 sz,..1 ..,. .J-...z.
Agitation 101711 DEX-HPLC was performed for all the 20 rnM phosphate formulations at all conditions The main peak percentage for the TO control at pH 7O with 0%, 0.02%, 0.04%, and 0.06% PS-80 was 68.88%, 69.83%, 71.36%, and 70.38%, respectively (Table 29). At 0.04% PS-80, the main peak percentage for the TO control at pH 6.5, 7.0, and 7.5 was 70.11%, 71.36%, and 68.04%, respectively. No significant change in the IEX
profile of all formulations was observed after exposure to freeze/thaw or agitation stress.
These results indicate little benefit in the addition of PS-80 up to 0.06% wiv.

"fable 29 Cotttritimi*At iigesegg,,,.7.,=-,7:7::n=,R....7,77:;!,ntr..:77..7,..E??4.giiiiifiiiiigi:
,immmm mmv:,,qmummmmmmmmn]m],?:.õ.:.y,.::m]ummmtIFn!ivnmRt;o115cmomm%...,:.;,3*.:.:,::
:]umi;ii ! TO 1 11.78 19.15 68,88 19.34 1 FIT 10.35 19.14 68.98 20.87 3 FIT 10,01 19_13 69.24 2075.
0,00%
48H Contrzai 10.12 19.10 68.72 21.17 24H Agitation 9.47 19_10 69.36 21,27 48H Agitation 9.44. 19.08 69.26 21,30 TO 9,06. 19.08 69.83 21.12 1 FIT 8.77 '19.07 70.44 20.78 3 FIT 8_38 19.07 70.66 20.96 0.021.'4) 481-1Controi 9.04 19.07 69,87 21.09 24H Agitatiori 9.35 19.07 69.73 20,92 7.0 48H Agitation 9.13 19.08 69,85 21.02 TO 8,89 19.08 71.36 19.75 1 FIT 6,97 19.07 70.67 20.36 3 FIT 8.98 19.07 70.94 20,08 0.04%
48H Control 9.23 19.07 70,87 19.90 24H Agitation 8.93 19.08 71..24 19,84 48H Agitation 9,38 19.08 70.52 20.09 TO 8.67 19.08 70.38 2075.
1 FIT 9,20 19.09 70.85 20.15 a FIT 9.86 19.08. 69,60 ,-,n .....,--=,...,,,.:1-,-....
0.08%
48H Control 9,55 19.08 69.41 21.04 24H Agitation 9.57 19_08 69.53 20.91 48H ;Agitation 9.33 19.06 69.01 21,12 TO 7.15 19,08. 70,11 20.84 1 FIT 8.98 19.08 70,16 20.86 3 FIT 9,29 19_09 69.64 21.07 6.5 48H Control 9.50 19.09 69,35 21.25 24H Agitation 9,23 19.06 69.67 21.11 48H Agitation 9.78 19.08 68õ93 21,30 0.04%
TO 11.32 19.08 69.04 20.84 1 FIT 10.78 19.08. 68,65 20.56 3 FIT 10.70 19.08 68.79 20.51 7..5 481-4 Control 10,91 19.09 68.07 21.02 24H Agitation 10.57 19.08 69.05 20.38 48H Agitation 10.32 19.03 68.18 21.00 Example 4: Salt/Trehalose Dependence Study 101721 The preformation salt/trehalose dependence study performed at 22 mg/mL
L-asparaginase was formulated in nine different formulation buffers, according to Table 30.
An aliquot of 27 inL of Lot RE-LAP-P59 was divided into 6 portions of approximately 3 mL
(3 portions) and 6 m-L (3 portions). A buffer exchange was performed for formulations 1-6 via dialysis using 10K MWCO Slide-A-Lyzer G2 Cassettes. Dialysis was performed at room temperature at a minimum buffer to sample volume ratio of 40:1. Four dialysis buffer exchanges were performed for each formulation approximately every 2 hours, with one exchange occurring overnight Table 30 "i:iiiti- 164. 34ft4:P1kMZMtaflktiC4tltOSVSM
amm zgairohltimm Fl 0 6mL
20mM r.la F2nmL
Phosphate F3. 100 b rni_ F4 3mL
M 0m Na F5 175 od 3mL
Phosphate F7 M 0 No DWysis 20m Na F8 002 No Dat'lisis Phosphate F9 100 No 'Daysis 101731 Following dialysis, the material was removed from the dialysis cassettes and the pH and protein concentration were confirmed, The concentration of the samples was then adjusted to 20 2 mg/mL with the corresponding buffer. After adjustment, the material in buffers 1-3 was split into equal portions of 3mL each. One set of these aliquots was supplemented with 10% PS-80 to a final concentration of 0.02%. All samples were then filtered and vialed in a biosafety cabinet. One set of the formulations were stored at 5 C until testing for the initial material quality evaluation, one set was stored at 5 C
for 3 weeks, and one set was subjected to a heat stress at 40 C for 3 weeks. The 3 week time point was selected after an evaluation of the visual appearance for after 1.5 weeks storage at 40 C.
Formulations one and two at 5 C were additionally tested by 11-X and SEC FIPLC
after seven weeks.
101741 The salt/trehalose dependence study was performed to assess the impact of sodium chloride concentration on L-asparaginase formulations in 20 mM or 50 milvl phosphate, 175 mIVI trehal ose, at pH 7.0 with and without 0.02% PS-80 (Table 30). The formulations were exposed to 1.5 and 3 weeks at 5 C and 40 C Size exclusion and 1EX
chromatography were used to assess the stability of RC in the presence of these varying salt concentrations in the selected buffers. Osmolality and conductivity were also determined at the initial timepoint and are shown in Table 31 Table 31 BuffPr Exciplent" excizient 2 PS-80 > Osnx,..faIrty tendue.1.1%.eits?
(mSicrn) 240 2.3 20 .mk1 Na 50 rnMNaCi :333 7 Ph.f3s.phate 100 rM NC i 425 7 c rntiol Na 175 rrifk-I 7 0 4'31 0 Phosphi30. Trehah)se 100 mt,INaC",1 =$:4 20 er ki Na mk4 NaCr; 7 1 Phsptiat=F
100 FTIM NaCi 002 431 Z1 101751 By appearance testing, all initial and stressed samples were colorless and slightly opalescence with very-few to no product related particulates. All formulations became more opalescent as a Function of stress. The 0.02% PS-80 containing formulations did not have any particulates present regardless of storage duration (Table 32) This result indicates that addition of 0.02% PS-80 may delay particle Formulation during long term storage. There was no dependence of appearance on salt concentration. The protein concentration was also assessed for initial and stressed formulations The protein concentration was between 19.7 and 25.2 mg/mL (Table 33) No significant changes or trends were observed in the stressed samples n >
o u..
NJ
I-.
I, --.1 NJ

NJ

NJ
L.' ,.
NJ
J
V
M

(1,I0 N
N
N
--,..
N
I-, .-1 N
Co) . , I-, .g . .iff.,A,.....::::::-....f:itiotTfir,!.......,Ek.fmrerrt %..... ''. 44 :, 7'..7.$1,11t .p..0,._,:=:=.=== ,..., ,...., 'fi, ':=::
:=,;:i...,a,..,;:, . f, ':, ... ... ........... ...
....44,,,,,43,,,. .. ... .... .... õ......, , ........ = -a: ff.. "47.7i 77.7i7::::::'''ilkJialit.'=47:::::: .. -,,.i4 ,....: '...,...; = = ..... l= = ....... = = = = = ,2 .7:7 i,..' so :::,...,:: = sii,..
:70cov,i.:.47,.. ..... :, ........ sn,.. -447.7,4rw:m.: ..... .
...::.,..74=.:. . ...37:7 ..7 ::::: ::::::: :7:7*7..ii ..7:7:7*
::::::: ::::::::?7µ..arn4.:47:7:7: .7:7',.., ::::::: ;ix .:,..f.,' .
............ ........ .......... ..õ......
TC: iNiw, =7:7cW3:144ss, 'Lleuir.1 Clere7.- Lio7.4f.: FR4e ,4r %=::::.'le = .'f,'N 45.-C
C):4oriess:LIqurri Opaiesoence 3 ocrl mere rri-orramme than Reference Suspension N Free ....-.1Vise F,ales 3ce,,5ess14,33im 7,-40,:rtresce.line ,,s. 7-4-4 more pronry.in4;>e, frion Relererr4e Su.spensio_in N Free of e,,S:ble PallaPat.E.IS

47C CP;odess:Liqtricl QpaleSfrenOS: :a no3 n4nre pro;r,orsce. Irian Reteremce Suspension N Very Few Palionlales 75 NA Co4orresr,L17:517,-.1 Clear Lig7ii.,:l Free of: vlsrl-le Ral[CO2,1eS.
.20 3417.4 Na 55 roM li 5W 43`C
Cof4r14,,ss1_174747.71 ppMeSnerine ',...'...; F3 frrare pronourr. Mon Refere.737:e Suspension ir,r- Free of -',"3-s3e. Pate3raters Pnrasprrate iflaC',' ---- .5'.1.,:=
Cnkyless .Ll--qui,c, r_alesc=ence.,s r.23 3115r.e 23e0543n5e than Reflerence Suspension N Free 7.-a'3s441e Paetane.3., 43'S .Ccfartess1_1333377-3 Ormt7escenne "4-7. F.,,7. more pronouno?, Mon Refere7Ke Suspension 3'4' 'Very Few Ponloulales TS NA =53323ss l_lqufri Dear L:iguld Free 7:r73,7kutile ParlicUates 11,-,0. nif,A liW 43...,õ
C:eMriese -L17-4:47,-1 Opalescence is nr-.7% worerri-orreunce trran Reference Sue:pension P.i Free r..1vis":331e PaRicufales IlaC'',' -- 1..5W C,-.4oriess:LOo3 OpaleSfenCe a .17c% Friffe rIFOrt<TWiFef_:e. till3n Reference Sec-1.-persion l7.,' Free ..-3f. =4s"..3e P7-37lic33e."3 .-3W 43'=5:
0147-17ess=LIn3371 DpMesnenne is nol frx-ye proname.e. Mari Refere7re S'osf-..serson IV Ven.= Few NiNes TS NA Cr:Ad:less 'Licrtri,1 Cle:ar Licari. Free 13risr14le Pf37-3774Liar, -7.3W -/D'e 0447:Mes5 =Domni 5sprI7eszence is r23 MOre prrinoLnore Man Referenne Susrfentnon "XI
FfÃE' d: V.8',...1".4e Peitiag3erS
---i--, 175 ral __________________________________ :3Vi 43`33.
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'';:': Fief: Cg'47f,Tle PatiC.1geS
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Free at visitle 77,n3073,3:_es n Cl) N

N
N
=-....

.-1 I-, !A
cn N

Table 33 - :i- - . . : , PS46", = ,,, -1.
Ayeracie"......'''''.11 Exien E.xciplent . Ilme - :
4:ormulation Buf ciptt fer '',1,1 condition ConcontratiorV

TO WA
28.4 24.

40x::.

TO WA

20 fziMila Phospiniate F2 50 n-i:M Nai:-. S5C
2 .6 40-,:i 27.8 TO WA
23.7 100 rriM NaC.'i ----.:,,,,y 40 C.
..-.:,.-.; 9 TO WA
22.4 2iN 40N._.'.

175 rail __ Trehalos, TO WA

'Hi il-tM Na F5 58 rnivi NiaU
Pilosphate 3Vi 4,>=1'C

TO WA
21.7 p$3 1430 rars4NaC.::
SW 40c 25 .-,s F7 ---- 0.02 :iilAi 40'.1.z.
24.2 TO NA
22.5 20 rnM Na FR .50 rrthl NaC o.e2 Picisspliate 3Vi 4O'C

TO WA

190 rnM NoC1 882 SW 4C_N:;

WC)2022/217231 F17171US2022/071562 [0176] IEX-IIPIA: results at the initial time point showed similar profiles with main peak percentages between 64.1% and 66.4%, acidic species percentages between 5.9%
and 7.5%, and basic species percentages between 27.7% and 28.7%. After three weeks at 40 C the samples showed significant differences in charge heterogeneity profiles with main peak percentages between 35.0% and 42.3%, acidic species percentages between 22.5% and 32.0%, and basic species percentages between 33.0% and 36.2%. After 7 weeks at 5 C only slight variation was observed with main peak percentages between 59.3% and 61.3%, acidic species percentages between 9.7% and 12.8%, and basic species percentages between 27.9%
and 29.0% (Table 34). Formulations with salt have slightly higher main peak percentages, compared to formulations without salt. Addition of 0.02% PS-80 had little to no benefit in terms of charge heterogeneity.
Table 34 4"."""""""..:4""-Aõ,,.1""..,....,...õ,a,----V.*-Z,;.,,' "Excipient Excipin:l. '''. Time ..' ' -"1'7 ii --'"'7 =::..
iiiiPiarnluation :Suffer = 80 % Concllia3) Acidic Main Peak . i. 1 2 : . -Point = ..ii .iiiMp:SPecies= C:41,:,::. =Ni .:::::+:Species i'.4::::, TO t4,'A 1 7,49 64,1-. 29.42 F ;=5-:=., 12.76 59.3:6 '7,94 ?,1N
4t1 C, 26.84 33.24 35.11 2Ct gaol N-3 3-C WA 6.31 65.51 25,17 F-2 v:lospizaze 971 51 4a.f: 26,94 36.04 35.11 IE3r,M TI WA 6.06 65.65 2608 NaCi 3W 4'C 2:3.26 40,61 36.13 TI WA 8.33 66.58 27.99 -;75 inNI, 4.fi'f: 22.46 41.39 36.16 Mehaiose _______________________ - -1.-' NIA 5.85 66.42 27.72 if.ci into tics " iiim FF, Piv3sphaie Nit,,:: S1W ,1;C 26.87 40.32 33.91 IOC ril M 1';_ ;s4,=,% 5.99 58.32 27.69 NaC: SW 46C 24.02 41.99 33,99 TO 13A 8.42 65.37 28.21 F7 0.52 3W 46 24,47 42.27 33.26 2'3 raM Na 9C; 4-,,M TI NeA 8.11 65.42 26,48 3W 4f 1 .97 35.03 93.08 ',Naci' ci ri., I'M it, M -T-) N,';-; 6.13 65.21 25.66 46W. 29.40 39.23 35.98 ' Sampies v.,..isre not analyzed until 7 ,eiiis after the en.idy i.,:es initiated.
[0177] SEC41PLC results at the initial time point showed similar profiles with main peak percentages between 98.0% and 98.2% andiliMW peak percentages between L9%
and 2.0%. After three weeks at 40 C the samples showed slight degradation in purity due to the appearance of aLMW peak. The main peak percentages for the three week stressed samples were between 94.8% and 95.4%, with HMNA" peak percentages between 1.9%
and 2.1% and LlvliNV peak percentages between 2.7% and 3.1%. After three weeks at 5 C no significant changes in main peak and 1-1N1W peak percentages were observed (Table 35).

High and low molecular weight species are minimized with addition of salt, and no benefit was observed when the formulation was supplemented with 0.02% PS-80.
Table 35 '........:.................:E............14...c-;p-e-n-t.:E-x-elpR" ' r :-H
MW Pe ;::-;""""........;;;:;:;::.;.........'......;;;:;:-:
:;.].;.:.;:.......7....;;;.:
'Rale ,i*,-,,-,- :-:,*:- ak -:-:-triam Peak* ::iii:1--l'Ait,i Peak BU x L1 ii:i . i 80,%.. point 4% I'44 ]]]
0,:;3 TO NIA 1,05 99.15 ---F 1 .5 C 1.85 9.6.15 2.11 9.1.79 3.10 20 rref.4 No T,.3 N,'A 1,87 98.13 ---F2 Flit.whalk, 5(2, nIN1 1.84 _;:
4C,C, 1.83 55.28 2.36 I Cr, :TIM -f-D WA' 1.9C-A 98.10 1.55 95.19 2.96 1.98 98.02 ---175 rnr,1 4c,,N7. 2.02 95.15 2.83 5,2 mf.4 Ma Trtthalw-''' .5,7, mr,1 F5 -F3 *A 1 .97 92.03 ---Ftvyapnote ' 1.95 95.28 2.75 FEi 1,-..,--,, 1.95 95.05 ---N;s1Ci .]:`ei 4;-.0 1.92 95.40 2.88 FT 1.93 93.07 ---II: 02 2.13 94.82 F 2:_l ITIM N13 tA3 ,-n?.,1 -$-Q N.:A
1.93 m.07 ---a o :32 FnasisNne NaC.: `2,15'1 2. C.C. 1.90 95.14 2.96 ICC rn N1 TO 0 02 r.VA 1.87 48.18 ---Fg 1.89 95.19 2.91 Example 5: Comparison between Recombinant Crisantaspase derived from Pselidomonas (RC-P) and Erwinase Experiments were performed to compare the relevant quality attributes of Recombinant Crisantaspase derived from P seudomonas (RCP) and Erwinase. A
summary of the data and results are presented in Table 36, comparing the RC-.P to Erwinase to demonstrate comparability. The results demonstrate that the RCP and Erwinase are comparable in their structures and enzymatic activities. Size and charge differences were observed.
Table 36: Summary of Comparability Results Attribute Method RC-P Erwinase Results Peptide Confirms the expected primary Comparable, with Primaty Map LC- - amino acid sequence, with very glycation observed on Structure MS consistent PTMs lysine for Erwinase --------------------------------------------------------------------------------- -Comparable Monomer Molecular weight Intact Mass Confirms the expected measurement, (denatured) monomer molecular weight glycation observed in Erwinase HMW:0.2% HMW:7.3%
Comparable profiles and main contents;
SE-IIPLC Main: 99.6% Main: 92.3%
Erwinase has higher LMW: 0.1% LMVV: 0.3%
HMW content Size Comparable profiles and tetramer SEC- Tetrarner: Tetrarner:
dominates, Erwinase MALLS 99.6% 93.3%
has higher octamer content _______________________________________________________________________________ ____ ---1 Comparable profiles Tetramer (with and tetramer dominant, SEC-MS Tetramer:
glycation) Erwinase has glycation (Native) 99.7%
92.7% and higher level of octatners CIEX Acidic:11% Acidic:18%
Comparable profiles Main:84% Main:75%
Erwinase has higher (CIEX-MS) Basic: 5% Basic: 7%
acidic variants Charge pI of main: 8.6 pI of main: 8.6 Acidic:15% A.cidic:46% Comparable main peak iCI:EF
Main:74% Main:45%
pI Erwinase has higher acidic variants Basic: 11% Basic: 8%
RP-UTIPLC Prepeak: 14.1% Prepeak: 17.2%
(RP- Main:83.2% Main:81.2%
Comparable profiles UHPLC- Postpeak: l'ostpeak:
and main contents MS) 2.7% 1.5%
Hydrophobicity Comparable HIC
Main: 94% profiles and main Main: 98% Minor 2%
contents Erwinase has Minor 2% Late eluting late eluting peak which peak: 4% correlate to HMW
species.
Very consistent deuterium Higher Order Comparable higher I-IDX MS uptake maps for RC-P and Structure order structures Erwinase Nessler Potency Activity 685 U/mg 572 U/mg Comparable Assay 101791 The peptide mapping MS data was mined for peptides containing common or known modifications including oxidation, deamidation, succinimide, isomerization, glycation, methylation and acetylation. The relative extent of each modification was calculated using counts for modified peptides normalized against total peptides, modified and unmodified. The relative level reported here was semi-quantitative.
Methylation and acetylation modifications were not found in any of the materials. Oxidation, deamidation and succinimide were found in both RC-P and Erwinase. Their levels were comparable and relatively low as shown in Table 37 (Note: PTMs lower than 0.5% are not reported). In addition, low level of glycations were observed in multiple lysine positions in Erwinase only.
Additional glycation modifications in Erwinase would affect its charge properties, resulting in increase of its acidic variants. Overall, both RC-P and Erwinase have confirmed primary amino acid structure. Their PTMs are comparable, except the additional glycation modification observed in Erwinase.
Table 37: Observed Post-translational Modifications Post-translational Modification RC-P Erwinase Deamidation % (N281) 2.0 2.3 Oxidation % (M133) 0.5 0.7 Succinimide (N281) 3.9 4.7 Glyeation% (K3 or K53) ND 2.0 Glyeation% (K219) ND 0.8 Glyeation% (1(269) ND 2.4 Glyeation% (K318) ND 1.7 [0180] Size-exclusion Chromatography (SE-LI:HP:LC) provides quantitative information about the molecular size distribution of a native protein. The SE-UHPLC
profiles for the RC-P and Erwinase showed relative peak area distribution of size-exclusion peaks as listed in Table 38. Both RC-P and Erwinase had dominant main peak content. The amount of high-molecular-weight species (H:MW) observed for Erwinase (7.3%) was higher than RC-P (0.2%). The amount of low-molecular-weight species (LMW) observed for both RC-P (0.1%) and Erwinase (0.3%) was comparable.
Table 38: Percent HMW, Main Peak and LMW from SE-UHPLC Analysis Sample ID/area% HMW2 HMW1 Main Peak LMW
RC-P ND 0.2 99.6 0.1 Erwinase 1.9 5.4 92.3 0.3 Size Exclusion Chromatography with Multi angle Laser Light Scattering:
SE-HPLC MALLS were performed for both RC-P and Erwinase. The identification of size-exclusion peaks was listed in Table 39. SE-I-PLC separation combined with MALLS
performed on both RC-P and Erwinase identified the SEC main content as tetramer, with a molecular weight in the range of 133-134 kDa. Both materials showed dominant tetramer content. HMW1 species for both materials was identified as the octamer with a MW in the range of 277-286 kDa, which was close to the theoretical octamer mass of 280 kDa. 1-11V1W2 species observed only for Erwinase was identified as the hexadecamer (16-mer) with a MW
of 554 kDa, which matched to the theoretical hexadecamer mass of 560 kDa. The size of LMW species could not be accurately assessed by MALLS due to the very low abundance of these forms in both RC-P and Erwinase. SEC-MALLS results showed that both RC-P
and Erwinase contained dominant tetramer form, their ITMW forms were similar, identified as octarners. In addition, Erwinase contained low level of 16-mer.
Table 39: SEC-MALLS Results RC-P Erwinase Peak/sample/ID
Measured Measure MW
MW Da Identity Da Identity HMW2 NA NI) 554.1 Hexadecamer (16-mer) BMW! 277.5 Octamer 286.1 Octamer Main 133.2 Tetramer 133.9 Tetramer LMW NA NA NA NA
101821 Imaged Capillary Isoelectric Focusing (iCIEF): iCIEF is an orthogonal assay to provide quantitative measurement of the charge heterogeneity of RC-P
protein. The iCIEF profiles for RC-P and Erwinase, relative peak areas for the main peak, acidic group and basic group with respect to the total area were listed in Table 40. Both materials showed highly comparable pI values for the main content. Erwinase had higher acidic variants content.
Table 40: iCIEF Charge Variants Distribution for RC-P and Erwinase PI Percent CYO
Sample Name Acidic Main Peak Main Peak Basic Variants Variants RC-P 8.6 15 74 11 Erwinase 8.6 46 45 8 Note: The total % may not add exactly to 1000/0 due to rounding.
Oxidation study:
101831 Both RC-P and Erwinase samples were subjected to forced oxidation by treatment with 0.01% (v/v) H202 for 4 h at room temperature, respectively.
Analytical testing results showed that the oxidized RC-P and Erwinase samples had around 30%
oxidation of methionine residues in total, about 20% increase in pre-peak contents of RP-UHPLC, an increase in acidic variants of iCIEF, a slightly increase in UMW content of SEC and had comparable CIEX, HIC, activity properties. Detailed analytical results are discussed below.
101841 Peptide Mapping Results: The tryptic peptide mapping LC-MS methods were employed to confirm the primary structure of the protein and identify potential post translational modifications induced by forced oxidizing conditions, especially methionine oxidations. Peptide mapping results showed that the forced oxidized RC-P and Erwinase had the expected primary structure with oxidization of methionine at multiple locations (Table 41). Overlays of the peptide mapping profiles of H202 treated RC-P and Erwinase are comparable. Both oxidized sample traces shown an increase peak intensity of oxidized peptide containing M133, M60 and M121 respectively. The H202 treated RC-P and Erwinase showed different levels of oxidation at multiple methionine locations (Table 41).
The methionine locations prone to oxidation from the most to least susceptible were: M133 (-.20%)> M60 (--.8%)> MI2 I (-4%) > M308 (-1%). A.round 33% total methionine oxidation was observed for both samples. Similar levels of deamidation and succinimide intermediates at Asn281 were observed for both oxidized samples and the reference standard.
Table 41: PTMs for the Oxidized RCM' and Erwinase Post-translational Erwinase RC-P RS RC-P Oxidized Modification Oxidized Deamidation % (N281) 2.8 2.8 3.3 Oxidation % (M133) 0.5 19.5 18.5 Oxidation % (M60) 09 I
7.7 Oxidation % (MI21) 0.7 4.3 4.2 Oxidation % (M308) 0.6 i. 1.2 1.1 Succinimide 'Vo (N281) 4.5 4.6 4.5 Glycation% (1(3/1(30/ K53) ND ND
3.4 Glycation% (K265/K269) ND ND
2.3 Glycation% (K318) ND ND
1.4 [0185] SE-UHPLC Results: The SE-UHPLC method was used to evaluate the size distribution of RC-P in its native state under nondenaturing conditions. The overlays of the oxidized RC-P, oxidized Erwinase and reference standard were analyzed. There were no significant profile differences observed between the oxidized samples and the RS. Both oxidized samples showed predominately tetrarner peak, a slightly increase of the HMW
contents and comparable LMW contents, compared to the reference standard. The relative peak intensities are listed in Table 42.

Table 42: SE-IMPLC Results Sample ID/area% HMW I /HMW2 Main Peak LMW
RC-P RS (RM-M-0.2 99.6 0.1 0009) Erwinase 7.3 92.3 0.3 Erwinase Oxidized 11.7 88.2 0.1 RC-P (GM1)4) 1.2 98.7 0.1 Oxidized 101861 CIEX Results: The CIEX method was used to evaluate the charge distribution of RC-P in its native state under non-denaturing conditions. The overlays of the oxidized RC-P, oxidized Erwinase and the reference standard were analyzed.
There were no significant profile changes between the oxidized samples and the reference standard. Both of the oxidized samples showed predominately main peak content. The relative peak intensities of the stressed samples and reference standard are listed in Table 43.
Table 43: CIEX Results Sample ID/area% Acidic% Main%
Basic%
RC-P RS (RM-M-0009) :Erwinase 18 75 7 Erwinase oxidation 14_:) 72 9 stressed RCP (GM:P4) oxidation stressed 101871 iCIEF Results: The iCEEF method is an orthogonal method to evaluate the charge distribution of RC-P with an applied electrical field. The overlays of the oxidized sample and the reference standard were analyzed. Their relative peak intensities are listed in Table 44. Both oxidized samples showed an increased acidic peak at p1 around 8.5.
Oxidation have no impact for the basic variants.
Table 44: iCIEF Results for the Oxidized Sample and RS
Sample 1D/area% Acidic A Main % Basic %
RC-P RS (RM-M-

14 73 12 0009) Erwinase 46 45 8 Erwinase oxidation stressed GMP4 oxidation stressed Low pH study:
101881 Both RC-P and Erwinase samples were subjected to low pH treatment with 50mM sodium phosphate at pH 3.4 at room temperature, then frozen at -80 C. Pre-study showed that the incubation time at pH 3.4 was not critical, from 15 min up to 7 days, the SE-UHPLC profiles of treated samples were comparable. Treated samples were relatively stable stored at -80 C up to 8 weeks. Analytical testing results showed that the treated RC-P and Erwinase samples had around 10% monomer content with reduced activities. Their peptide mapping profiles, PTM, CIEX, RP-UHPLC and HIC properties were comparable to the untreated. Detailed analytical results are discussed below.
[0189] SE-UHPLC Results: The SE-LTFEPLC method is used to evaluate the size distribution of RC-.P in its native state under nondenatufing conditions. The overlays of the low pH treated RC-P, Erwinase and reference standard were analyzed. Both low pH treated RC-P and Erwinase showed predominately tetramer peak and around 10% increasing in ILMW contents, compared to the reference standard. Low pH treatment led to slightly increase of HMVV content for Erwinase from 7.3% to 10.6% while no impact for RC-P. Their relative peak intensities are listed in Table 45.

Table 45: SE-UHPLC Results Sample 1D/area% HMW1/HMW2 Main Peak LIVINV
RC-P RS (RM-M-0.2 99.6 0.1 0009) Erwinase 7.3 92.3 0.3 Erwinase low pH
10.6 79.9 9.5 treated RC-P (GMIN) low 0.2 87.4 124 pH treated CIEX Results: The CIEX method is used to evaluate the charge distribution of RC-P in its native state under non-denaturing conditions. The overlays of the low pH treated RC-P. Erwinase and the reference standard were analyzed. There were no significant profile changes between the pH treated samples and the reference standard. Both of the treated samples showed predominately main peak content. The relative peak intensities of the treated samples and reference standard are listed in Table 46.
Table 46: CEEX Results Sample Iniarea% Acidic% Main% Basic%
RC-P RS (RM-M-0009) Erwinase 18 75 7 Erwinase low pH

treated RC-P (GMP4) low pH treated 101911 iCIEF Results: The iCIEF method is an orthogonal method to evaluate the charge distribution of RC-P with an applied electrical field. The overlays of the low pH
treated RC-P, Erwinase and the reference standard were analyzed. Their relative peak intensities are listed in Table 47. Both low pH treated RC-P and Erwinase showed an increased acidic peak at pl around 7.2, which was identified as monomer previously. Low pH
treatment had no impact for the basic variants.
Table 47: iCIEF Results for the Oxidized Sample and RS
Sample 1D/area% Acidic A Main % Basic %
RC-P RS (RM-M-0009) Erwinase 46 45 8 Erwinase low pH

treated RC-P (GMP4) low pH treated 101921 Comparability of RC-P and Erwinase was evaluated by comparing their physicochemical, purity, structural and potency characteristics. Size-based heterogeneity, analyzed by SE-UHPLC, SEC-MALLS and SEC-MS, is comparable. Both materials have dominant tetramer structure. The HMW species was identified as an octamer.
Erwinase has higher levels of octamer. Low levels of 16-mer species were also observed in Erwinase (<2%). It has previously been demonstrated through size variant characterization of RC-P
that the octamer shows comparable activity with tetratner. Overall, the observed size distribution differences between Erwinase and RC-P has minimum impact on drug efficacy.
Erwinase has glycation modifications, which contribute to the acidic variants differences. The higher level of octamer/16-mer observed in Erwinase contributed to the late eluting basic variants shown on CIEX. Previous forced degradation study of RC-P demonstrates the glycated RC-P have comparable structure and activity to RC-P. Hydrophobic profiles, as measured by /11C and RP-UHPLC, are comparable for both materials. Minor differences were observed are due to the glycation modification and higher level of HMW
content of Erwinase. Primary structure and higher order structure of both materials are comparable.
Potency results are comparable. Forced degradation study indicates that Erwinase and RC-P
have comparable stability under oxidized and low pH stress. Both oxidized RC-P
and Erwinase with around 20% increasing in pre peak of RP-UTIPLC have comparable activity compared to their untreated materials. Both RC-P and Erwinase treated with low pH stress condition have reduced activity, which correlates with the monomer contents of the treated samples very well. Based on these data, RC-P demonstrate comparable structure, biological properties and function to Erwinase.
ENUMERATED EMBODIMENTS
101931 The following enumerated embodiments are representative of some aspects of the inventions.
101941 Embodiment 1: An aqueous, non-lyophilized formulation, comprising:
(i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1;
(ii) one or more disaccharides, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof; and (iii) one or more buffers, wherein the one or more buffers are substantially free of amino acid, wherein the formulation comprises less than about 5% low-molecular-weight (LMW) species after storage at 37 C, for one week.
101951 Embodiment 2: The formulation of embodiment 1, wherein the L-asparaginase is present at a concentration of about 20 mg/mL.
101961 Embodiment 3: The formulation of embodiment 1 or embodiment 2, wherein the L-asparaginase is non-PEGylated and non-PASylated.
101971 Embodiment 4: The formulation of any one of embodiments 1-3, wherein the one or more disaccharides comprise trehalose.
101981 Embodiment 5: The formulation of embodiment 4, wherein the trehalose is present at a concentration of between about 50 mlµ4 and about 300 mM.

101991 Embodiment 6: The formulation of embodiment 4, wherein the trehalose is present at a concentration of between about 150 mM and about 275 mM.
102001 Embodiment 7: The formulation of embodiment 4, wherein the trehalose is present a concentration of about 170 mM.
102011 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.
102021 Embodiment 9: The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise a phosphate buffer.
102031 Embodiment 10: The formulation of any one of embodiments 1-7, wherein the one or more buffers comprise sodium phosphate.
102041 Embodiment 11: The formulation of embodiment 10, wherein the sodium phosphate is present at a concentration of between about 0.5 mM and about 50 mM.
102051 Embodiment 12: The formulation of embodiment 10, wherein the sodium phosphate is present at a concentration of about 20 mM.
102061 Embodiment 13: The formulation of any one of embodiments 1-12, wherein the formulation further comprises sodium chloride.
102071 Embodiment 14: The formulation of embodiment 13, wherein the sodium chloride is present at a concentration of between about 25 mM and about 150 mM.
102081 Embodiment 15: The formulation of embodiment 13, wherein the sodium chloride is present at a concentration of about 50 mM.
102091 Embodiment 16: The formulation of any one of embodiments 1-15, wherein the formulation further comprises one or more excipients.
102101 Embodiment 17: The formulation of embodiment 16, wherein the one or more excipients comprise polysorbate 80.

102111 Embodiment 18: The formulation of embodiment 17, wherein the polysorbate 80 is present a concentration of between about 0.004% (w/v) and about 0.2%
(w/v).
102121 Embodiment 19: The formulation of embodiment 17, wherein the polysorbate 80 is present at a concentration of about 0.02% (w/v).
102131 Embodiment 20: The formulation of any one of embodiments 1-19, wherein the formulation has a pH of between about 4.0 and about 8.5.
102141 Embodiment 21: The formulation of any one of embodiments 1-19, wherein the formulation has a pH of about 7Ø
102151 Embodiment 22: A method of treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject a formulation of any one of embodiments 1-21.
102161 Embodiment 23: The method of embodiment 22, wherein the disease, condition, or disorder is cancer.
102171 Embodiment 24: The method of embodiment 23, wherein the cancer is acute lymphoblastic leukemia (ALL).
102181 Embodiment 25: The method of embodiment 24, wherein the ALL is relapsed ALL.
102191 Embodiment 26: The method of embodiment 23, wherein the cancer is lymphoblastic lymphoma (LBL).
102201 Embodiment 27: The method of embodiment 26, wherein the LBL is relapsed LBL.
102211 Embodiment 28: The method of any one of embodiments 22-27, wherein the formulation is administered intramuscularly.
102221 Embodiment 29: The method of any one of embodiments 22-27, wherein the formulation is administered intravenously.

102231 Embodiment 30: The method of any one of embodiments 22-29, wherein the formulation is co-administered with one or more other chemotherapeutic agents.
102241 Embodiment 31: A kit, comprising: (i) a formulation of any one of embodiments 1-21; and (ii) instructions for treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof 102251 Embodiment 32: The kit of embodiment 31, wherein the disease, condition, or disorder is cancer.
102261 Embodiment 33: The kit of embodiment 32, wherein the cancer is acute lymphoblastic leukemia (ALL).
102271 Embodiment 34: The kit of embodiment 33, wherein the ALL is relapsed ALL.
102281 Embodiment 35: The kit of embodiment 32, wherein the cancer is lymphoblastie lymphoma (LBL).
102291 Embodiment 36: The kit of embodiment 35, wherein the LBL is relapsed LBL.

Claims

PCT/US2022/071562What is claimed is:

1. An aqueous, non-lyophilized formulation, comprising:
(i) an L-asparaginase, wherein the L-asparaginase comprises four monomer units, wherein each monomer unit has an amino acid sequence that is at least about 70% identical to SEQ ID=NO: 1; and (ii) one or more stabilizers, or one or more buffers, or any combination thereof.

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

3. The formulation of claim 2, wherein the one or more disaccharides comprise trehalose, sucrose, or any combination thereof.

4. The formulation of any one of claim 1 to 3, wherein the one or more buffers are substantially free of amino acid.

5. The formulation of any one of claim 1 to 4, wherein the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 40 C for two months.

6. The formulation of any one of claim 1 to 4, wherein the formulation comprises less than about 0.6% low-molecular-weight (LMW) species after storage at 37 "C for one week.

7. The formulation of any one of claim 1 to 6, wherein the forrnulation comprises less than 2% high-molecular-weight (UMW) species after storage at 40 "C for two months.

8. The formulation of any one of claim l to 7. wherein the 1...-asparaginase is present at a concentration of about 20 mg/mL.

9. The formulation of any one of claim 1 to 8, wherein the L-asparaginase is non-PEGylated and non-PASylated.

10. The formulation of any one of claim 1 to 9, wherein the one or more disaccharides comprise trehalose.

11. The formulation of any one of clairns 1-10, wherein the one or more buffers comprise a phosphate buffer, an acetate buffer, or any combination thereof.

12. The formulation of any one of claims 1-11, wherein the one or more buffers comprise a phosphate buffer.

13. The formulation of any one of claims 1-12, wherein the one or more buffers comprise sodium phosphate.

14. The formulation of claim 13, wherein the sodium phosphate is sodium phosphate dibasic anhydrous, sodium phosphate monobasic rnonohydrate, or a combination thereof.

15. The formulation of claim 13, wherein the sodium phosphate is present at a concentration of between about 0.5 mM and about 50 mM.

16. The formulation of any one of claims 1-15 wherein the formulation further comprises sodium chloride.

17. The formulation of claim 16, wherein the sodium chloride is present at a concentration of between 25 mM and about 150 mM.

18. The formulation of any one of claims 1-17, wherein the formulation further comprises one or more excipients.

19. The formulation of claim 18, wherein the one or more excipients comprise polysorbate 80, polysorbate 20, poloxamer 188, or any combination thereof.

20. The formulation of claim 18, wherein the one or more excipients are present at a concentration of between about 0.004% (w/v) and about 0.3% (w/v).

21. The formulation of any one of claims 1-20, wherein the formulation has a pH of between about 4.0 and about 8.5.

22. A rnethod of treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof, comprising administering to the subject a formulation of any one of claims 1-21.

23. The method of claim 22, wherein the disease, condition, or disorder is cancer.

24. The method of claim 23, wherein the cancer is acute lymphoblastic leukemia (ALL).

25. The method of claim 24, wherein the ALL is relapsed ALL.

26. The method of claim 23, wherein the cancer is lymphoblastic lymphoma (LBL).

27. The method of claim 26, wherein the LBL is relapsed LBL.

28. The method of any one of claims 22-27, wherein the forrnulation is administered intramuscularly.

29. The method of any one of claims 22-27, wherein the formulation is administered intravenously.

30. The method of any one of claims 22-27, wherein the formulation is administered subcutaneously.

31. The method of any one of claims 22-30, wherein the formulation is co-administered with one or more other chemotherapeutic agents.

32. A kit, comprising: (i) a formulation of any one of claims 1-21; and (ii) instructions for treating a disease, condition, or disorder that is treatable by asparagine depletion in a subject in need thereof.

33. The kit of claim 32, wherein the disease, condition, or disorder is cancer.

34. The kit of claim 33, wherein the cancer is acute lymphoblastic leukemia (ALL).

35. The kit of claim 34, wherein the ALL is relapsed ALL.

36. The kit of claim 33, wherein the cancer is lymphoblastic lymphoma (LBL).

37. The kit of claim 36, wherein the LBL is relapsed LBL.

38. A unit dosage form, comprising: (i) a formulation of any one of claims 1-21; and (ii) one or more pharmaceutically acceptable excipients.