Formulations of polyalkylene oxide-asparaginase and methods of making and using the same

Abstract

Embodiments of the invention include polyalkylene oxide-asparaginase compositions.SOLUTION: In an example, the composition is a lyophilised storage stable composition. In some examples, the lyophilized composition comprises one or more of a buffer, a salt, and a sugar. Embodiments of the invention further include methods of making the compositions. The compositions find use in a variety of applications, such as, for example, treating a neoplastic condition in a subject.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE AND INCORPORATION BY REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 62 / 344,249, filed June 1, 2016, U.S. Provisional Patent Application No. 62 / 344,252, filed June 1, 2016, and U.S. Provisional Patent Application No. 62 / 344,256, filed June 1, 2016, each of which is incorporated by reference in its entirety. [Background technology]

[0002] [First] L-asparaginase is an enzyme that hydrolyzes the amino acid L-asparagine via a deamination reaction to produce L-aspartic acid and ammonia. The enzymes include two types of asparaginase isozymes, L-asparaginase I and L-asparaginase II. L-asparaginase I is present in the cytosol, and asparaginase II is present in the cytosol. However, L-asparaginase II has low affinity for peri-asparaginase. It exists in the plasma and has a high affinity for L-asparagine. E. coli L-asparaginase II is a tetramer of identical subunits. , also known as L-asparagine amide hydrolase, type EC-2, EC 3.5.1.1.

[0003] L-asparaginase is known to have therapeutic value against leukemia. L-asparaginase is an aminohydrolase that catalyzes the reaction of L-asparagine to L-aspartic acid and ammonia. It is a soluble form of L-asparagine in plants, animals, and microorganisms. Currently, the therapeutic activity of this enzyme is being investigated by the It has been established that this condition is caused by the depletion / removal of circulating L-asparagine, an essential nutrient for the growth and survival of tumor (leukemia) cells with impaired asparagine synthesis. The ability to synthesize L-asparagine is not impaired in normal cells.

[0004] Administration of L-asparaginase to leukemia patients induces selective death of tumor cells by hydrolysis of L-asparagine, resulting in the treatment of malignant tumors. In some cases, L-asparaginase itself suffers from the typical disadvantages of protein therapeutics, such as high clearance and the potential for immune responses in patients treated with this enzyme. To address these deficiencies, polyethylene glycol-conjugated derivatives of L-asparaginase (PEG-asparaginase) have been developed. L-asparaginase II extracted from Escherichia coli can be used to produce P EG-asparaginase can be produced, which is substantially non-antigenic and This indicates a decreased rate of clearance from the circulation.

[0005] PEG-asparaginase liquid injection formulation (Oncaspar®) is already available in the U.S. It has been approved for commercial marketing by the US Food and Drug Administration. Oncaspar® is approved as a component of first-line multi-agent chemotherapy regimens for patients with acute lymphoblastic leukemia (ALL). In addition, Oncaspar® is approved for the treatment of ALL patients who are hypersensitive to asparaginase (e.g., the native form of L-asparaginase). is approved for. Summary of the Invention

[0006] Embodiments of the present invention include polyalkylene oxide-asparaginase compositions. In some instances, the compositions include one or more buffers and salts. In other embodiments, the compositions are frozen. The freeze-dried composition is a dried, shelf-stable composition. In some instances, the freeze-dried composition includes one or more buffers, salts, and sugars. Embodiments of the present invention further include methods for making the composition. The composition finds a variety of uses, such as, for example, in treating a neoplastic condition in a subject. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows a process flow diagram for a method of making a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 2] FIG. 2 shows a graph of purity (%) versus time (weeks) at 40° C. for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 3] FIG. 3 shows a graph of potency (IU / mL) versus time (weeks) at 40° C. for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 4] FIG. 4 shows a graph of purity (%) versus time (weeks) at 25° C. for a lyophilized storage-stable composition according to an embodiment of the present disclosure. [Figure 5] FIG. 5 shows a graph of potency (IU / mL) versus time (weeks) at 25° C. for a lyophilized shelf-stable composition according to an embodiment of the present disclosure. [Figure 6] 6 shows a flow diagram of a process for making a lyophilized, shelf-stable composition according to an embodiment of the present disclosure. The final formulation and filtration steps are shown. [Figure 7] 7 shows a flow diagram of a process for making a lyophilized shelf-stable composition according to an embodiment of the present disclosure. The aseptic filling and lyophilization steps are shown. [Figure 8] FIG. 8 shows a graph of purity (%) by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored at 2-8°C (e.g., 5°C). [Figure 9]FIG. 9 shows a graph of potency (activity) (IU / mL) versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored at 2-8°C (e.g., 5°C). [Figure 10] FIG. 10 shows a graph of total aggregate by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored at 2-8° C. (e.g., 5° C.). [Figure 11] FIG. 11 shows a graph of purity (%) by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under accelerated conditions (25±3° C.; 60%±5% RH). [Figure 12] FIG. 12 shows a graph of potency (activity) (IU / mL) versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under accelerated conditions (25±3° C.; 60%±5% RH). [Figure 13] FIG. 13 shows a graph of total aggregate by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under accelerated conditions (25±3° C.; 60%±5% RH). [Figure 14] FIG. 14 shows a graph of purity (%) by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under heat stress conditions (40±2° C.; 75%±5% RH). [Figure 15] FIG. 15 shows a graph of potency (activity) (IU / mL) versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under heat stress conditions (40±2° C.; 75%±5% RH). [Figure 16] FIG. 16 shows a graph of total aggregates by GF-HPLC versus time (months) for lyophilized compositions according to embodiments of the present disclosure stored under heat stress conditions (40±2° C.; 75%±5% RH). DETAILED DESCRIPTION OF THE INVENTION

[0008] [Definition] In describing the embodiments of the present disclosure, the following terms will be used and as indicated below: is intended to be defined as

[0009] "Substantially purified" means containing a majority of the sample in which a substance was present. For example, a substantially purified sample may contain 50% or more of the desired substance, such as 60% or more of the desired substance, such as 75% or more of the desired substance, such as 90% or more of the desired substance, such as 95% or more of the desired substance, including 99% or more of the desired substance. Any convenient procedure can be used to purify the desired substance, and includes, but is not limited to, filtration (e.g., defiltration, ultrafiltration, etc.), selective precipitation, crystallization, ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0010] "Isolated" means that the compound of interest is in an environment other than that which the compound naturally occurs in. "Isolated" means that the compound of interest in a sample is substantially enriched and / or that the compound of interest has been purified or substantially purified.

[0011] The terms "patient" and "subject" are used interchangeably and refer to a living organism that is suffering from or prone to suffering from a condition that can be prevented or treated by administering the compositions of the present disclosure, including both humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other primates and monkeys; livestock animals such as cows, sheep, pigs, goats and horses; domesticated mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domestic fowl, wild and game birds such as chickens, turkeys and other poultry birds, ducks, geese and the like. This term does not denote a specific age. Thus, adult, juvenile and newborn individuals are included.

[0012] "Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound or composition sufficient to treat a particular disease or disorder or one or more symptoms thereof and / or prevent the disease or disorder. With respect to a neoplastic condition, a pharmaceutically or therapeutically effective amount refers to, inter alia, an amount sufficient to cause a reduction in the amount and / or incidence of cancer in a patient and / or to reduce the rate of cancer growth.

[0013] As used herein, the term "treating" or "treatment" means treating or curing a disease or medical condition in a patient, such as a mammal (e.g., a human), and includes: (a) preventing the occurrence of a disease or medical condition, such as prophylactic treatment of a subject; (b) alleviating a disease or medical condition, such as causing elimination or reduction of a disease or medical condition in a patient; (c) inhibiting a disease or medical condition, for example, by slowing or halting the progression of a disease or medical condition in a patient; or (d) alleviating the symptoms of a disease or medical condition in a patient.

[0014] The term "physiological conditions" is meant to encompass conditions compatible with living cells, for example, predominantly aqueous conditions of pH, salinity, compatible with living cells.

[0015] Before the embodiments of the present disclosure are described in detail, it should be understood that the embodiments may vary and are not limited to the specific embodiments described herein. It should also be understood that the terminology used herein is used for the purpose of describing specific embodiments only and is not intended to be limiting. The scope of the embodiments of the present disclosure is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a range of values ​​is provided, any intervening values ​​between the upper and lower limits of the range are also included unless the context clearly dictates otherwise. It is understood that all stated or intervening values ​​in a stated range, to the nearest tenth of the lower limit, are encompassed within the embodiments of the present disclosure. The upper and lower limits of these smaller ranges are independently included in the smaller ranges and are also encompassed within the embodiments of the present disclosure, subject to the specific excluded limit from the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention. Certain ranges herein are expressed by numerical values ​​prefixed with the term "about." As used herein, the term "about" provides a literal aid for providing a number that is close to or approximately the exact number preceding it. For purposes of determining whether a number is near or approximating a recited number, the near or approximating number provides the substantial equivalent of the particular recited number in the context in which it is expressed.

[0016] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each listed publication, patent, and patent application is incorporated herein by reference to disclose and describe the subject matter for which the publications are cited. The cited publications are provided solely for their disclosure prior to the filing date of the present application and should not be construed as an admission that the invention herein is not entitled to antedate such publication by reason of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be individually confirmed.

[0017] It should be noted that the claims may be drafted to exclude any optional elements. Such statements are intended to serve as predicate basis for the use of the exclusionary terms "solely," "only," etc. in connection with claim elements, or the use of "negative" limitations. As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete elements and features that are readily separable from, or combined with, elements and features of other embodiments without departing from the scope and spirit of the embodiments of the present disclosure. Any recited method can be carried out in the order of events recited or in any order that is logically possible. Any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of embodiments of the present disclosure. Representative illustrative methods and materials are disclosed.

[0018] [Detailed Description of the Invention] Embodiments of the present invention include polyalkylene oxide-asparaginase compositions. In some instances, the compositions include one or more buffers and salts. Embodiments of the present invention further include methods of making the compositions. The compositions find a variety of uses, such as, for example, in treating neoplastic conditions in a subject.

[0019] In other embodiments, the composition is a lyophilized, storage-stable composition. Embodiments of the invention include a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. In some examples, the lyophilized composition includes one or more buffers, salts, and sugars. Embodiments of the invention further include methods of making the composition. The composition finds a variety of uses, including, for example, treating a neoplastic condition in a subject (e.g., acute myeloid leukemia (AML) in a subject).

[0020] Aspects of the invention include administering an effective dose of polyalkylene oxide-asparaginase. Aspects of the invention further include compositions and kits containing polyalkylene oxide-asparaginase that can be used to treat a subject.

[0021] In further describing embodiments of the present disclosure, compositions (e.g., liquid and lyophilized) will first be described in detail, followed by methods of making and using the compositions and kits containing the compositions.

[0022] [Composition] Aspects of the present disclosure include compositions of polyalkylene oxide-asparaginase, which include a polyalkylene oxide group covalently bonded to the asparaginase via a linker. The compositions can also include one or more buffers and salts. In certain embodiments, the compositions are lyophilized, storage-stable compositions. The lyophilized, storage-stable compositions can also include one or more salts and sugars.

[0023] As described herein, the compositions of the present disclosure can include polyalkylene oxide-asparaginase. Polyalkylene oxide-asparaginase includes asparaginase covalently bound to one or more polyalkylene oxide groups via a linker. Asparaginase is an enzyme composed of four identical subunits, with one active site per tetramer. For example, the asparaginase enzyme can be L-asparaginase (e.g., For example, L-asparaginase II), which reacts with amino acids according to the following reaction: L-asparagine ((S)-2,4-diamino-4-oxobutanoic acid, or asparagine) (also known as (S)-2-aminosuccinic acid) and ammonia from acetylcholinesterase (also known as acetylcholinesterase, abbreviated as Asn or N): [ka]

[0024] In some cases, asparaginase catalyzes the synthesis of the amino acid L-glutamylase according to the following reaction: L-glutamic acid (also known as (S)-2-aminopentanedioic acid, abbreviated as Gln or Q) is produced from 2,5-diamino-5-oxopentanoic acid (also known as (S)-2,5-diamino-5-oxopentanoic acid, abbreviated as Gln or Q). (which is produced by the reaction of acetone with acetone) and ammonia: [ka]

[0025] The above reaction mediated by L-asparaginase is also called a deamination reaction. In one example, the L-asparaginase in the composition may be derived from, but is not limited to, E. coli ( The asparaginase may be derived from a prokaryotic source, such as a microorganism containing an asparaginase-expressing asparaginase (e.g., E. coli asparaginase). In this case, the asparaginase in the composition of interest is E. coli asparaginase. In some cases, the asparaginase is expressed by E. coli. The asparaginase is recovered and purified from a medium containing the asparaginase-expressing E. coli. In addition to wild-type asparaginase, the asparaginase can also be a non-naturally occurring asparaginase and / or a synthetically produced asparaginase and / or an active fragment of a natural and / or synthetic asparaginase. Asparaginases that can be used in embodiments of the present invention include, but are not limited to, those described in the following: 9,322,008; 9,127,266; 9,051,561; 8,617,868; 7,807,436; 6,991,788; 6,537,547; 6,436,396; 6,368,845; 6,274,367; 6,251,388; 6,165,735; 6,140,101; 6,087 ,151; 6,042,825; 5,854,051; 5,310,670; 4,729,957 and 4,617,271; the disclosures of which are incorporated herein by reference.

[0026] As described above, the asparaginase in the polyalkylene oxide-asparaginase composition is an asparaginase covalently bound to one or more polyalkylene oxide groups. For example, the asparaginase may include asparaginase covalently bound to one or more polyalkylene oxide groups in a post-translational process. The polyalkylene oxide-asparaginase may include asparaginase covalently bound to one or more polyalkylene oxide groups at one or more positions of the asparaginase. For example, the polyalkylene oxide group can be covalently bound to an amino acid residue of the asparaginase. In some cases, the polyalkylene oxide group can be covalently bound to the amino group of an amino acid residue of the asparaginase. In some embodiments, the polyalkylene oxide group is covalently bound to the amino acid side chain of the N-terminal amino acid of the asparaginase. In some embodiments, the polyalkylene oxide group is covalently attached to the epsilon-amino group of a lysine (K) in the asparaginase. In some embodiments, the polyalkylene oxide group is covalently attached to the amino acid side chain of the N-terminal amino acid of the asparaginase and to the epsilon-amino group of a lysine (K). In some cases, the polyalkylene oxide-asparaginase is substantially non-antigenic. "Non-antigenic" or "substantially non-antigenic" means that the composition does not elicit a significant immune response when administered to a subject. In some examples, the polyalkylene oxide-asparaginase has a reduced clearance rate from the circulation of a subject compared to unmodified asparaginase. For example, the elimination half-life of the polyalkylene oxide-asparaginase can be 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, 18 days or more, 19 days or more, 20 days or more, etc. In some embodiments, the elimination half-life of the polyalkylene oxide-asparaginase is 3 days or more. In some embodiments, the elimination half-life of the polyalkylene oxide-asparaginase is 5 days or greater.

[0027] The polyalkylene oxide group attached to the asparaginase can be any physiologically acceptable polyalkylene oxide group. Poly(alkylene oxide)s (PAOs), also known as polyoxyalkylenes (POAs), are synthesized by the polymerization of alkylene oxides (e.g., ethylene oxide, propylene oxide, butylene oxide). Homopolymers are synthesized from only one type of alkylene oxide, while copolymers are synthesized from two or more different alkylene oxides and are known as alkylene oxide copolymers (AOCs). Examples of the former are poly(ethylene oxide) (PEO), a polymer of ethylene oxide (EO), and poly(propylene oxide) (PPO), a polymer of propylene oxide (PO). Poly(ethylene oxide) is also commonly known as polyethylene glycol (PEG) or polyoxyethylene (POE). The molecular weight of such polymers is generally characterized as the average length (or repeat unit). In addition to the standard linear shape, branched or star-shaped poly(alkylene oxides) are also available, each of which can serve as an initiation point for growing polymer chains. They can be synthesized by initiating the polymerization reaction with a multifunctional initiator containing multiple hydroxyl, amino, or thiol groups. For example, using glycerin (with three hydroxyl groups) as an initiator results in a three-arm branched polymer, while pentaerythritol results in a four-arm polymer. Traditionally, polymers of this type with three to ten arms are called "branched," while those with ten or more arms are called "star" polymers. "Comb" copolymers are similar to branched and star copolymers, except that the initiators in comb copolymers each function as the initiation point for growing polymer chains. "Graft" copolymers are multifunctional polymers in which multiple hydroxyl, amino, or thiol groups are located along the backbone of the initiator, allowing for the addition of pendant polymer chains along the polymer backbone with unsaturated C=C bonds, or the pendant chains are bonded to the polymer backbone by reaction. They can be synthesized using monofunctional polymer chains with pendant functional groups (e.g., hydroxyl groups) that can be added. All poly(alkylene oxide)s contain, in addition to the repeating units derived from alkylene oxide, a single residue corresponding to the molecule used to initiate the polymer synthesis. For linear polymers, the alkylene oxide corresponding to the alkylene oxide used in the synthesis glycols (e.g., ethylene glycol and ethylene oxide, respectively) and therefore the residue derived from the initiator is indistinguishable from other repeating units of the polymer chain. Small molecules other than alkylene glycols are often used as initiators, examples of which are methanol or N-butanol (for linear polymers) and trimethylolpropane. Examples of initiators include propane, glycerol, and pentaerythritol (for branched polymers) or ethylenediamine. The mass of the initiator relative to the mass of the final polymer chain is generally very small and usually negligible. Thus, the term poly(alkylene oxide) is used herein in its conventional sense and includes both poly(alkylene oxides) initiated from alkylene glycol molecules and poly(alkylene oxides) initiated from other small molecules.

[0028] In certain embodiments, a physiologically acceptable polyalkylene oxide group is substantially stable under conditions compatible with living cells, e.g., temperatures, pHs, and salt concentrations that are compatible with living cells, mostly aqueous conditions. The group is water soluble. The term "water soluble polymer" refers to a polyalkylene oxide group that is substantially soluble in water, such as the aqueous conditions found in a subject's body. Polyalkylene oxide groups of interest include, but are not limited to, linear polyalkylene glycols. Linear polyalkylene glycols used in certain embodiments of the present invention are of the following formula: [ka]

[0029] wherein R is selected from the group consisting of hydrogen, lower alkyl, and mixtures thereof; R is selected from the group consisting of hydrogen and lower alkyl; and n is a positive integer. "Lower alkyl" means alkyl having 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, butyl, and isomers thereof. R can be selected from the group consisting of hydrogen, methyl, and mixtures thereof; R can be selected from the group consisting of hydrogen and methyl; and n can be a positive integer selected to provide the desired polymer size.

[0030] In certain examples, the poly(alkylene glycol) used in embodiments of the present invention is poly(ethylene glycol), poly(propylene glycol), mixtures thereof, and copolymers of poly(ethylene glycol) and poly(propylene glycol), wherein one of the terminal hydroxyl groups of the polymer may be substituted with a lower alkyl group. In some embodiments, the polyalkylene oxide is polyethylene glycol (PEG. In particular embodiments, the polyethylene glycol (PEG) has a molecular weight of 1,000 to 20,000 daltons. In particular embodiments, the PEG has a molecular weight of 1,000 to 20,000 daltons, or 1,000 to 19,000 daltons, or 1,000 to 18,000 daltons, or 1,000 to 17,000 daltons, or 1,000 to 16,000 daltons, or 1,000 to 15,000 daltons, or 1,000 to 14,000 daltons, or 1,000 to 13,000 daltons, or 1,000 to 12,000 daltons. The PEG has a molecular weight of 2,000-10,000 daltons, or 1,000-11,000, or 1,000-10,000, or 1,500-10,000 daltons, or 2,000-10,000 daltons, or 2,000-9,000 daltons, or 2,000-8,000 daltons, or 2,000-7,000 daltons, or 2,000-6,000 daltons, or 3,000-6,000 daltons, or 4,000-6,000 daltons, or 4,500-5,500 daltons. In certain embodiments, the PEG has a molecular weight of 2,000-10,000 daltons. In certain embodiments, the PEG has a molecular weight of 4,000-6,000 daltons. In certain embodiments, the PEG has a molecular weight of 5,000 daltons. In one example, the polyethylene glycol is a methoxypolyethylene glycol (eg, monomethoxypolyethylene glycol, or "mPEG").

[0031] As described above, the polyalkylene oxide group can be covalently attached to the asparaginase. In some cases, the polyalkylene oxide group is covalently attached to the asparaginase via a linker. In this case, the polyalkylene oxide group can be covalently attached to the asparaginase via a linker. The linker can be any convenient linker that allows for the attachment of the polyalkylene oxide group to the asparaginase. For example, the linker can include a reactive functional group that provides a covalent bond between the polyethylene oxide group and the asparaginase. In some cases, the linker includes a reactive functional group that provides a covalent bond between the polyethylene oxide group and an amino acid of an amino acid residue of the asparaginase. For example, the linker can include a reactive functional group that provides a covalent bond between the polyethylene oxide group and the amino group of an amino acid residue of the asparaginase. Examples of such reactive functional groups include, but are not limited to, p-nitrophenoxy, thiazolidinyl thione, N-hydroxybenzoates, and the like. Roxisuccinimidyl, or, but not limited to, N-hydroxybenzotriazolyl , halogen, N-hydroxyphthalimidyl, imidazolyl, O-acylurea, pentafluorophenol, or 2,4,6-trichlorophenol, or other suitable reactive functional groups. In some cases, the reactive functional group of the linker is N-hydroxysuccinimidyl. Thus, for covalently attaching the polyalkylene oxide group to the asparaginase, the linker may include, but is not limited to, functional groups such as a urethane linker (also known as a carbamate linker), a succinic acid linker, and the like.

[0032] In certain embodiments, the linker comprises a urethane linker (also known as a carbamate linker). For example, the attachment of methoxypolyethylene glycol (mPEG) to the amino group of an amino acid of a polypeptide (e.g., asparaginase) via a urethane (carbamate) is shown below. [ka]

[0033] In the reaction shown above, methoxypolyethylene glycol succinimidoyl carbonate (also referred to as SC-PEG) is attached to the carboxyl group of a polypeptide (e.g., asparaginase). SC-PEG-asparaginase reacts with the amino group of amino acids to produce polyethylene glycol-asparaginase with a urethane (carbamate) linker. llo, AL., et al., “Pharmacokinetic (PK) and pharmacodynamics (PD) properties of SC-PEG E. coli 1-asparaginase (EZN-2285) in the O ncology Group (COG) study AALL07P4”, 2012 A American Society of Clinical Oncology (ASCO) ) Annual Meeting, Poster 9543; and Angiolillo, AL., et al., "Pharmacokinetic and Pharmacodynamic Properties of Pegaspargase (Pegol E) scherichia coli L-Asparaginase in the Trea tment of Patients With Acute Lymphoblastic Leukemia: Results From Children's Oncology Group Study AALL07P4”, J Clin. Oncology, 3 2(34),2014,3874-3882.

[0034] In certain embodiments, the linker is a succinic acid linker (also called a succinyl linker). For example, the attachment of methoxypolyethylene glycol (mPEG) to an amino group of an amino acid of a polypeptide (e.g., asparaginase) via a succinic acid linker is shown below. [ka]

[0035] In the reaction shown above, methoxypolyethylene glycol succinimidyl succinate (also called SS-PEG) is attached to the amino acid residue of a polypeptide (e.g., asparaginase). It reacts with the amino group of the acid to form polyethylene glycol-asparagine with a succinic acid linker. SS-PEG-asparaginase is also described in U.S. Patent Nos. 5,122,614; 5,324,844; and 5,612,460, the disclosures of each of which are incorporated herein by reference.

[0036] In certain embodiments, the composition comprising the polyalkylene oxide-asparaginase is a dehydrated composition. As used herein, a dehydrated composition has a pH of 25% or less, or 20% or less, by Karl Fischer (KF) titration. A dehydrated composition is a composition containing a low amount of water, such as 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less water. In some cases, the dehydrated composition contains 3% or less water by Karl Fischer titration. In some cases, the dehydrated composition contains 1% or less water by Karl Fischer titration. In some cases, the dehydrated composition contains 0.5% or less water by Karl Fischer titration. Any convenient procedure can be used to produce the dehydrated composition, such as increasing the temperature of the composition (e.g., heating), reducing pressure, lyophilization (also known as freeze-drying), etc., and combinations thereof.

[0037] In certain embodiments, lyophilization is used to produce a dehydrated composition, and thus the composition (e.g., a composition comprising polyalkylene oxide-asparaginase) is a lyophilized composition. In some examples, a lyophilized composition is one in which water has been removed from the composition by sublimation, where the water in the composition undergoes a phase transition from solid to gas. For example, a lyophilized composition can be one in which water has been removed by freezing the composition (e.g., freezing the water in the composition) and then reducing the pressure around the composition such that the water in the composition sublimes. As noted above, a lyophilized composition may have a water content of 25% or less, or 20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% or less, as measured by Karl Fischer titration. The lyophilized composition may contain a moisture content of 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less, or 0.5% or less, or 0.25% or less, or 0.1% or less, etc. In certain embodiments, the lyophilized composition may contain a low moisture content, such as about 0.1% to about 25%, or about 0.25% to about 20%, or about 0.5% to about 15%, or about 1% to about 10%, or about 2% to about 9%, or about 3% to about 8%, or about 4% to about 7%, or about 5% to about 6%, as measured by Karl Fischer titration. In embodiments, the lyophilized composition may contain a low moisture content, such as about 0.1% to about 5%, or about 0.25% to about 4%, or about 0.5% to about 3%, or about 1% to about 2%, as measured by Karl Fischer titration. In some cases, the lyophilized composition contains 3% or less water as measured by Karl Fischer titration. In some cases, the lyophilized composition contains 1% or less water as measured by Karl Fischer titration. In some cases, the lyophilized composition contains 0.5% or less water as measured by Karl Fischer titration.

[0038] Due to the low moisture content of the lyophilized composition, as described above, the lyophilized composition can be in a solid form. In some cases, a solid lyophilized composition is a powder. In some cases, the lyophilized composition facilitates storage of the composition for extended periods of time (e.g., compared to a liquid formulation of the same composition). For example, the lyophilized composition can be a storage-stable composition (e.g., a lyophilized storage-stable composition), which is substantially stable for extended periods of time. "Stable" or "substantially stable" or "storage-stable" refers to a composition that does not significantly degrade and / or lose activity over an extended period of time. For example, a storage-stable composition can contain no more than 10% impurities, or no more than 9%, or no more than 8%, or no more than 7%, or no more than 6%, or no more than 5%, or no more than 4%, or no more than 3%, or no more than 2%, or no more than 1%, etc., of significant impurities, such as degradation products, resulting from degradation of the composition, over an extended period of time. In certain embodiments, a storage-stable composition may have about 1% to about 10%, or about 2% to about 9%, or about 3% to about 8%, or about 4% to about 7%, or about 6% to about 5% or less of degradants over an extended period of time. In certain examples, a storage-stable composition has 5% or less impurities over an extended period of time. In some cases, a storage-stable composition substantially retains its activity over an extended period of time, such as retaining 100%, or 99% or more, or 98% or more, or 97% or more, or 96% or more, or 95% or more, or 94% or more, or 93% or more, or 92% or more, or 91% or more, or 90% or more, or 85% or more, or 80% or more, or 75% or more of its activity over an extended period of time. In some embodiments, the storage-stable composition substantially retains its activity over an extended period of time, such as retaining about 75% to about 100%, or about 80% to about 99%, or about 85% to about 98%, or about 90% to about 97%, or about 91% to about 96%, or about 92% to about 95%, or about 93% to about 94% or more of its activity over an extended period of time. For example, the storage-stable composition substantially retains 90% or more of its activity over an extended period of time.In some cases, a storage-stable composition substantially retains 95% or more of its activity over an extended period of time. An extended period of time is a period of time of 1 week or more, or 2 weeks or more, or 3 weeks or more, or 1 month or more, or 2 months or more, or 3 months or more, or 4 months or more, or 6 months or more, or 9 months or more, or 1 year or more, or 1.5 years (e.g., 18 months) or more, or 2 years or more, or 2.5 years (e.g., 30 months) or more, or 3 years or more, or 3.5 years (e.g., 42 months) or more, or 4 years or more, or 4.5 years (e.g., 54 months) or more, or 5 years or more. For example, an extended period of time can be 6 months or more. In some cases, an extended period of time can be 9 months or more. In some cases, an extended period of time can be 1 year (e.g., 12 months) or more. In some cases, an extended period of time can be 1.5 years (e.g., 18 months) or more. In some cases, an extended period of time can be 2 years (e.g., 24 months) or more. In some embodiments, an extended period of time is 6 months or more. The period can be from about 1 to about 3 weeks, or from about 1 month to about 6 months, or from about 6 months to about 9 months, or from about 1 year to about 1.5 years, or from about 1 year to about 2 years, or from about 1 year to about 3 years, or from about 1 year to about 4 years, or from about 1 year to about 5 years. In some embodiments, the storage-stable composition is substantially stable for extended periods at room temperature, such as temperatures of 20-40°C, or 25-35°C, or 25-30°C. In some examples, the storage-stable composition is substantially stable for extended periods at temperatures below room temperature, such as 0-20°C, 0-15°C, 0-10°C, or 2-8°C.

[0039] In some cases, the composition comprises a therapeutically effective amount of polyalkylene oxide-asparaginase, the enzymatic activity of which is measured in International Units (IU) and corresponds to the amount of enzyme required to produce 1 μmol of ammonia per minute at a pH of 7.3 and a temperature of 37° C. In some cases, the polyalkylene oxide-asparaginase can be present in the composition in an amount ranging from 100 to 5,000 IU / g (e.g., the polyalkylene oxide-asparaginase has potency (activity), such as 500 to 4,500 IU / g, or 500 to 4,000 IU / g, or 500 to 3,500 IU / g, or 500 to 3,000 IU / g, or 500 to 2,500 IU / g, or 500 to 2,000 IU / g, or 500 to 1,500 IU / g, or 500 to 1,000 IU / g, or 600 to 900 IU / g, or 700 to 800 IU / g. In a particular example, the polyalkylene oxide-asparaginase can be present in the composition in an amount of 500 to 1,000 IU / g. For example, the polyalkylene oxide-asparaginase is present in the composition at a potency (activity) of 500-1,000 IU / g. In particular examples, the polyalkylene oxide-asparaginase is present in the composition in an amount ranging from 700-800 IU / g. For example, the polyalkylene oxide-asparaginase is present in the composition at a potency (activity) ranging from 700-800 IU / g. In a particular example, the polyalkylene oxide-asparaginase is present in the composition in an amount of 750 IU / g. For example, the polyalkylene oxide-asparaginase may have a potency (activity) of 750 IU / g.

[0040] In some cases, a therapeutically effective amount of polyalkylene oxide-asparaginase is present in the composition, and the polyalkylene oxide-asparaginase is present in an amount of 55 IU / mg protein or more, or 60 IU / mg protein or more, or 65 IU / mg protein or more, or 70 IU / mg protein or more, or 75 IU / mg protein or more, or 80 IU / mg protein or more, or 85 IU / mg protein or more, or 90 IU / mg protein or more, or 95 IU / mg protein or more, or or 100 IU / mg protein or more, or 105 IU / mg protein or more, or 110 IU / mg protein or more, or 115 IU / mg protein or more, or 120 IU / mg protein or more, or 125 IU / mg protein or more, or 130 IU / mg protein or more, or 135 IU / mg protein or more, or 140 IU / mg protein or more, or 145 IU / mg protein or more, or 150 IU / mg protein or more. For example, the polyalkylene oxide-asparaginase in the composition can have a specific activity of 85 IU / mg protein or more. In some embodiments, the polyalkylene oxide-asparaginase in the composition has a specific activity of 50-150 IU / mg protein, or 55-145 IU / mg protein, or 60-140 IU / mg protein, or 65-135 IU / mg protein, or 70-130 IU / mg protein, or 75-125 IU / mg protein, or 80-120 IU / mg protein, IU / mg protein, or 95-105 IU / mg protein. In some cases, the polyalkylene oxide-asparaginase in the composition has a specific activity of 50-150 IU / mg protein, such as 65-140 IU / mg protein, or 70-135 IU / mg protein, or 75-130 IU / mg protein, or 75-125 IU / mg protein. For example, the polyalkylene oxide-asparaginase in the composition Polyalkylene oxide-asparaginase in the diet provides 75-125 IU / mg protein. The specific activity may range.

[0041] In certain embodiments, a therapeutically effective amount of polyalkylene oxide-asparaginase is present in the composition, and the polyalkylene oxide-asparaginase is present in the composition at a concentration of 1.5 mg / mL to 14.5 mg / mL, or 2 mg / mL to 14 mg / mL, or 2.5 mg / mL to 13.5 mg / mL, or 3 mg / mL to 13 mg / mL, or 3.5 mg / mL to 12.5 mg / mL, or 4 mg / mL to 12 mg / mL. In some cases, the alkylene oxide-asparaginase is present in the composition in an amount ranging from 1 mg / mL to 15 mg / mL, such as 4.5 mg / mL to 11.5 mg / mL, or 4.5 mg / mL to 11 mg / mL, or 4.5 mg / mL to 10.5 mg / mL, or 4.5 mg / mL to 10 mg / mL, or 4.5 mg / mL to 9.5 mg / mL, or 4.5 mg / mL to 9 mg / mL, or 4.5 mg / mL to 8.5 mg / mL, or 5 mg / mL to 8 mg / mL. There is.

[0042] When administered to a subject, the composition provides 500-5,000 IU / m 2 or 500-4,500 IU / m 2 , or 500-4,000 IU / m 2 , or 500 to 3,500 IU / m 2 or 500-3,000 IU / m 2 , or 1,000 to 3,000 IU / m 2 , or 1,500-3,000 IU / m 2 , or 1,750 to 3,000 IU / m 2 , or 2,000-3,000 IU / m 2 , or 2,000 to 2,750 IU / m 2 or 2,250–2,750 IU / m 2 Polyalkylene oxide-asparaginase, etc., 100 to 5,000 IU / m 2 Polyalkylene oxide-asparagine The composition can include a sufficient amount of polyalkylene oxide-asparaginase to deliver the enzyme to the subject. For example, the composition can include 1,500 to 3,000 IU / m 2 The composition may include an amount of polyalkylene oxide-asparaginase sufficient to deliver 2,000 to 2,750 IU / m of polyalkylene oxide-asparaginase to a subject. In a particular example, the composition may include 2,000 to 2,750 IU / m 2 The composition may include an amount of polyalkylene oxide-asparaginase sufficient to deliver 2,250 to 2,750 IU / m of polyalkylene oxide-asparaginase to a subject. 2 The composition may include an amount of polyalkylene oxide-asparaginase sufficient to deliver 2,500 IU / m of polyalkylene oxide-asparaginase to a subject. For example, the composition may include 2,500 IU / m 2 The polyalkylene oxide-asparaginase may comprise an amount of polyalkylene oxide-asparaginase sufficient to deliver to the subject.

[0043] In certain embodiments, the dosage form administered to the subject is a liquid dosage form, e.g., an aqueous dosage form. In some embodiments, in addition to the polyalkylene oxide-asparaginase, the dosage form includes a buffer and a salt.

[0044] The compositions of the present disclosure may contain additional components in addition to polyalkylene oxide-asparaginase. For example, the compositions may include a buffer. Buffers suitable for use in the compositions of the present disclosure include buffers that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable buffers include, but are not limited to, phosphate buffers (e.g., phosphate-buffered saline (PBS)), Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), Tris buffer, Ringer's lactate buffer, and the like, and combinations thereof. The buffer included in the composition can be a buffer that maintains the pH of the composition at a physiologically acceptable pH in the range of 6 to 8, or about 7, e.g., 7.2, 7.3, or 7.4. In one example, the buffer is a phosphate buffer. The buffer solution can include dibasic sodium phosphate (also known as disodium phosphate or sodium hydrogen phosphate; NaHPO) and / or can include monobasic sodium phosphate (also known as monosodium phosphate; NaHPO).

[0045] In some cases, the amount of dibasic sodium phosphate in the composition ranges from 0.05 to 5 wt.%, such as 0.1 to 4.5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3.5 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2.5 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1 wt.%, or 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.2 to 0.6 wt.%, or 0.3 to 0.6 wt.%, or 0.4 to 0.6 wt.%, or 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate can be present in the composition in the range of 0.1 to 1.0 wt.%. In certain examples, dibasic sodium phosphate can be present in the composition in a range of 0.2-0.8 wt.%. In certain examples, dibasic sodium phosphate can be present in the composition in a range of 0.3-0.6 wt.%. In certain examples, dibasic sodium phosphate can be present in the composition in a range of 0.5-0.6 wt.%. For example, dibasic phosphoric acid can be present in the composition at about 0.6 wt.%, such as 0.56 wt.% (or 0.558 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the composition is 0.01-1.8 wt.%, or 0.01-1.6 wt.%, or 0.01-1.4 wt.%, or 0.01-1.2 wt.%, or 0.01-1.0 wt.%, or 0.01-0.8 wt.%, or 0.01-0.6 wt.%, or 0.01-0.4 wt.%, or 0.0 The range is 0.005-2 wt.%, such as 1-0.2 wt.%, or 0.02-0.18 wt.%, or 0.03-0.16 wt.%, or 0.04-0.16 wt.%, or 0.045-0.15 wt.%, or 0.04-0.14 wt.%, or 0.05-0.14 wt.%, or 0.1-0.2 wt.%, or 0.1-0.15 wt.%. For example, monobasic sodium phosphate can be present in the composition in a range of 0.05-0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in a range of 0.01-0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in a range of 0.09-0.15 wt.%.In certain examples, monobasic sodium phosphate can be present in the composition in an amount ranging from 0.1 to 0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in an amount ranging from 0.1 to 0.15 wt.%. For example, monobasic sodium phosphate can be present in the composition in an amount of 0.12 wt.% (or 0.129 wt.%).

[0046] Another additional component that can be included in the composition of the present disclosure is a salt.Salts suitable for use in the composition of the present disclosure include salts that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, for example, by injection or intravenous administration.Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., and combinations thereof.In a specific example, the salt is sodium chloride.

[0047] In some cases, the amount of salt (e.g., sodium chloride) in the composition is in the range of 0.05 to 5 wt.%, such as 0.05 to 4 wt.%, or 0.05 to 3 wt.%, or 0.05 to 2 wt.%, or 0.1 to 5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1.5 wt.%, or 0.1 to 1 wt.%, or 0.2 to 1 wt.%, or 0.3 to 1 wt.%, or 0.4 to 1 wt.%, or 0.5 to 1 wt.%, or 0.6 to 1 wt.%, or 0.7 to 1 wt.%, or 0.8 to 1 wt.%, or 0.8 to 0.9 wt.%. For example, the salt (e.g., table salt) can be present in the composition in a range of 0.5 to 1 wt.%. For example, the salt (e.g., table salt) can be present in the composition in a range of 0.2 to 2 wt.%. In a particular example, the salt (e.g., table salt) can be present in the composition in a range of 0.5 to 1 wt.%. For example, salt (e.g., table salt) can be present in the composition in an amount ranging from 0.7 to 1 wt.%. In a particular example, salt (e.g., table salt) can be present in the composition in an amount ranging from 0.8 to 0.9 wt.%. For example, salt (e.g., sodium chloride) can be present in the composition in an amount of 0.85 wt.%.

[0048] In certain embodiments, the composition comprising polyalkylene oxide-asparaginase is a lyophilized composition. The lyophilized composition of the present disclosure may also comprise a buffer, a salt, and a sugar in addition to the polyalkylene oxide-asparaginase. For example, an embodiment of the present disclosure includes a lyophilized storage-stable composition of polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently bonded to the asparaginase via a linker, a buffer, a salt, and a sugar.

[0049] Suitable buffers for use in the lyophilized compositions of the present disclosure include those that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, such as by injection or intravenous administration. Examples of suitable buffers include those described above. In certain embodiments, the phosphate buffer may include dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the composition ranges from 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.1 to 0.4 wt.%, or 0.2 to 0.4 wt.%, or 0.2 to 0.3 wt.%, or 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in the composition in an amount ranging from 0.1 to 0.5 wt.%. In certain examples, dibasic sodium phosphate can be present in the composition in an amount ranging from 0.2 to 0.4 wt.%. In certain examples, dibasic sodium phosphate can be present in the composition in an amount ranging from 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in the composition in an amount of about 0.3 wt.%, such as 0.28 wt.% (or 0.279 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the composition ranges from 0.005 to 1 wt.%, such as 0.01 to 0.9 wt.%, or 0.01 to 0.8 wt.%, or 0.01 to 0.7 wt.%, or 0.01 to 0.6 wt.%, or 0.01 to 0.5 wt.%, or 0.01 to 0.4 wt.%, or 0.01 to 0.3 wt.%, or 0.01 to 0.2 wt.%, or 0.01 to 0.1 wt.%, or 0.02 to 0.09 wt.%, or 0.03 to 0.08 wt.%, or 0.04 to 0.08 wt.%, or 0.045 to 0.075 wt.%, or 0.04 to 0.07 wt.%, or 0.05 to 0.07 wt.%. For example, monobasic sodium phosphate can be present in the composition in an amount of 0.01-0.1 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in an amount of 0.05-0.07 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in an amount of 0.045-0.075 wt.%.For example, monobasic sodium phosphate can be present in the composition in an amount of 0.06 wt.%.

[0050] In certain embodiments, the lyophilized compositions of the present disclosure include salts. Suitable salts for use in the compositions of the present disclosure include salts that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, such as by injection or intravenous administration. Examples of suitable salts are described above. In a particular example, the salt is sodium chloride.

[0051] In some cases, the amount of salt (e.g., sodium chloride) in the composition ranges from 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.2 to 0.5 wt.%, or 0.3 to 0.5 wt.%, or 0.4 to 0.5 wt.%, or 0.4 to 0.45 wt.%. For example, the salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.1 to 1 wt.%. In certain examples, the salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.3 to 0.5 wt.%. In certain instances, a salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.4 to 0.45 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount of about 0.4 wt.%, such as 0.425 wt.%. Another component that can be included in the compositions of the present disclosure is a sugar. Suitable sugars for use in the compositions of the present disclosure include sugars that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable sugars include, but are not limited to, sucrose, mannitol, maltose, trehalose, and 2-hydroxypropyl-β-cyclodextrin (HPCD). , lactose, glucose, fructose, galactose, glucosamine, etc., and combinations thereof. In certain instances, the sugar is a disaccharide. For example, the disaccharide may be sucrose.

[0052] In some cases, the amount of sugar (e.g., sucrose) in the composition ranges from 0.1 to 25 wt.%, such as 0.5 to 20 wt.%, or 1 to 15 wt.%, or 1 to 10 wt.%, or 1 to 9 wt.%, or 1 to 8 wt.%, or 2 to 7 wt.%, or 2 to 6 wt.%, or 3 to 5 wt.%, or 4 to 5 wt.%. For example, the sugar (e.g., sucrose) can be present in the composition in an amount of 1 to 10 wt.%. In certain examples, the sugar (e.g., sucrose) can be present in the composition in an amount of 3 to 5 wt.%. In certain examples, the sugar (e.g., sucrose) can be present in the composition in an amount of 4 to 5 wt.%. For example, the sugar (e.g., sucrose) can be present in the composition in an amount of 4.5 wt.%.

[0053] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) in the range of 500-1,000 IU / g, dibasic sodium phosphate in an amount in the range of 0.1-1.0 wt.%, monobasic sodium phosphate in an amount in the range of 0.01-0.2 wt.%, salt (e.g., sodium chloride) in an amount in the range of 0.2-2 wt.%, and water.

[0054] In some embodiments, the composition comprises, consists essentially of, or consists of a polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount ranging from 0.2-0.8 wt.%, monobasic sodium phosphate in an amount ranging from 0.1-0.14 wt.%, a salt (e.g., sodium chloride) in an amount ranging from 0.6-1.0 wt.%, and water. In some embodiments, the composition comprises, consists essentially of, or consists of a polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount ranging from 0.5-0.6 wt.%, monobasic sodium phosphate in an amount ranging from 0.09-0.15 wt.%, a salt (e.g., sodium chloride) in an amount ranging from 0.09-0.15 wt.%, and water.

[0055] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, dibasic sodium phosphate in an amount of about 0.6 wt.%, monobasic sodium phosphate in an amount of about 0.1 wt.%, salt (e.g., sodium chloride) in an amount of about 0.9 wt.%, and water.

[0056] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, dibasic sodium phosphate in an amount of about 0.56 wt.%, monobasic sodium phosphate in an amount of about 0.13 wt.% (or 0.129 wt.%), salt (e.g., sodium chloride) in an amount of about 0.85 wt.%, and water.

[0057] In some embodiments, the composition comprises a polyalkylene oxide-asparaginase. , dibasic sodium phosphate, monobasic sodium phosphate, a salt (e.g., sodium chloride), and water.

[0058] In other embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase, dibasic sodium phosphate, monobasic sodium phosphate, a salt (e.g., sodium chloride), a sugar (e.g., sucrose), and water.

[0059] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) in the range of 500-1,000 IU / g, dibasic sodium phosphate in an amount in the range of 0.1-0.5 wt.%, monobasic sodium phosphate in an amount in the range of 0.01-0.1 wt.%, salt (e.g., sodium chloride) in an amount in the range of 0.1-1 wt.%, sugar (e.g., sucrose) in an amount of 1-10 wt.%, and water.

[0060] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount in the range of 0.2-0.4 wt.%, monobasic sodium phosphate in an amount in the range of 0.05-0.07 wt.%, salt (e.g., sodium chloride) in an amount in the range of 0.3-0.5 wt.%, sugar (e.g., sucrose) in an amount of 3-5 wt.%, and water.

[0061] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) in the range of 700-800 IU / g, dibasic sodium phosphate in an amount in the range of 0.25-0.3 wt.%, monobasic sodium phosphate in an amount in the range of 0.045-0.075 wt.%, salt (e.g., sodium chloride) in an amount in the range of 0.4-0.45 wt.%, sugar (e.g., sucrose) in an amount of 4-5 wt.%, and water.

[0062] In some embodiments, the composition comprises a polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, dibasic sodium phosphate in an amount of about 0.3 wt.%, monobasic sodium phosphate in an amount of 0.065 wt.%, salt (e.g., sodium chloride) in an amount of about 0.4 wt.%, sugar (e.g., sucrose) in an amount of about 4.5 wt.%, and water. Comprise, consist essentially of, or consist of.

[0063] In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 750 IU / g, dibasic sodium phosphate in an amount of about 0.28 wt.% (or 0.279 wt.%), monobasic sodium phosphate in an amount of 0.06 wt.%, salt (e.g., sodium chloride) in an amount of about 0.43 wt.% (or 0.425 wt.%), sugar (e.g., sucrose) in an amount of about 4.5 wt.%, and water.

[0064] In certain examples, the composition (e.g., a liquid or lyophilized composition) is a sterile composition. "Sterile" means that there are substantially no immunogenic components in the composition, e.g., substantially no pathogens (e.g., fungi, bacteria, viruses, spores, etc.). In some cases, the composition is present in a container. Providing a container for the composition facilitates maintaining the composition as a sterile composition. For example, the container can be configured to maintain the composition enclosed in the container in a sterile environment. As such, the container can be a sealed container, e.g., the container can have a watertight and / or airtight seal, etc. The seal may include a seal that can be removed to allow a user to access the contents of the container. In some instances, the seal may be a frangible seal, or in other instances, the seal may be removable by removing the seal from the container. The container may be configured to allow insertion of a needle, cannula, or syringe into the interior of the container without removing the seal. In some cases, a seal configured to allow access to the interior of the container without removing the seal from the container facilitates maintaining the contents of the container (e.g., the composition within the container) in a sterile environment prior to administering the composition to a subject. Suitable materials for the seal include, but are not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymer, polychloroprene, polyacrylic acid, and the like. Examples of suitable seals include rubber or polymer seals such as acrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. For example, in certain embodiments, the seal is a septum that can be penetrated by a needle, syringe, or cannula. The seal can provide convenient access to the sample in the container and also provide a protective barrier over the opening of the container. In certain examples, the seal is a removable seal, such as a screw-on or snap-on lid or other suitable sealing element that attaches to the opening of the container. For example, a screw-on lid can be screwed onto and removed from the opening before and after sample is added to the container.

[0065] In some cases, the container is a unit dose container. A unit dose container refers to a container containing one or more unit doses for administration to a subject. In some embodiments, the unit dose container contains a predetermined amount of a subject composition calculated to produce a desired effect in the subject, sufficient for the subject. Certain embodiments of the composition can be provided in a unit dose container suitable for individual administration of a precise dose. The amount of active composition administered to a subject can depend on the subject being treated, the severity of the affliction, and the mode of administration. For example, the unit dose container can contain an amount of a composition, as disclosed herein, in an amount sufficient to achieve a desired effect in the subject being treated. In certain examples, the unit dose container contains a composition having a therapeutically effective amount of polyalkylene oxide-asparaginase. Therapeutically effective amounts of polyalkylene oxide-asparaginase are described above. In certain embodiments, the unit dose container is a vial. In some cases, the vial is a sealed vial (e.g., as described above with respect to sealed containers).

[0066] The container can be constructed of any convenient material compatible with the polyalkylene oxide-asparaginase and other components of the composition. For example, the container can be a solid-compatible container configured to contain a solid (e.g., a lyophilized composition). In some instances, the container can be a liquid-compatible container configured to contain a liquid. The container can also be compatible with solids and liquids, where the container is configured to contain a solid and a liquid. In some instances, the liquid in the container can be an aqueous solution, and in these instances, the container is compatible with aqueous compositions. "Compatible" means substantially inert (e.g., does not significantly react with) the liquid and / or composition or other components that come into contact with the container. Examples of suitable container materials include, but are not limited to, glass and plastic. For example, the container can be constructed of, but is not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX TM The container may be made of glass, such as fused quartz glass, fused silica glass, etc. Other suitable materials for the container include, but are not limited to, plastics such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), polystyrene, etc. In a particular example, as described above, the container may be a vial, and in such a case may be a glass vial. As described above, the container may be a sealed container, The vial may be sealed and in such a case may be a sealed glass vial. As described in more detail below, liquid or reconstituted compositions of the present disclosure can be administered to a subject, for example, by injection or intravenously. In certain embodiments, prior to administering the reconstituted composition to a subject, the solid composition can be combined with a liquid, for example, as described above, to provide a liquid composition suitable for injection or intravenous administration. In some cases, prior to administering the composition to a subject, the solid composition can be combined with water (e.g., water for injection, WFI), for example, as described above, to provide a liquid composition suitable for injection or intravenous administration.

[0067] For example, a lyophilized composition can be reconstituted with water (e.g., water for injection, WFI) to produce a reconstituted dosage unit suitable for, e.g., injection or intravenous administration to a subject. As described herein, aspects of the present disclosure include compositions comprising: a polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently bonded to asparaginase by a linker; a buffer; and a salt. In certain embodiments, the polyalkylene oxide is polyethylene glycol, as described above. In certain embodiments, the linker is a urethane (carbamate) linker, as described above. In certain embodiments, the asparaginase is E. coli asparaginase, as described above. In certain embodiments, the buffer is a phosphate buffer, as described above. In certain embodiments, is sodium chloride, as described above. Thus, certain embodiments of compositions comprise polyethylene glycol-asparaginase comprising a polyethylene glycol group covalently bonded to E. coli asparaginase by a urethane linker; a phosphate buffer, and a salt. The individual components of these compositions (e.g., molecular weight of polyethylene glycol, amount of polyethylene glycol-asparaginase, type of phosphate buffer, and amount, type and amount of salt) are as detailed above.

[0068] As described herein, aspects of the present disclosure include a lyophilized, storage-stable composition comprising: a polyalkylene oxide-asparaginase comprising asparaginase covalently bound to a polyalkylene oxide group by a linker; a buffer, a salt, and a sugar. In certain embodiments, the polyalkylene oxide is polyethylene glycol, as described above. In certain embodiments, the linker is a urethane (carbamate) linker, as described above. In certain embodiments, the asparaginase is E. coli asparaginase, as described above. In certain embodiments, the buffer is a phosphate buffer, as described above. In certain embodiments, the salt is sodium chloride, as described above. In certain embodiments, the sugar is a disaccharide (e.g., sucrose), as described above. Thus, certain embodiments of the lyophilized, storage-stable composition include a polyethylene glycol-asparaginase comprising a polyethylene glycol group covalently bound to E. coli asparaginase by a urethane linker; a phosphate buffer, a salt, and a disaccharide. Each component of these compositions (e.g., molecular weight of polyethylene glycol, amount of polyethylene glycol-asparaginase, type and amount of phosphate buffer, type and amount of salt) Amounts) are as detailed above.

[0069] The compositions of the present disclosure may contain other components, such as additional pharmaceutically acceptable additives or excipients for dose delivery, as part of the composition. The additives may include, but are not limited to, carbohydrates, inorganic salts, organic salts, antimicrobial agents, antioxidants, surfactants, water (e.g., water for injection (WFI)), alcohols, polyols, glycerin, vegetable oils, phospholipids, buffers, acids, bases, and any combination thereof. Carbohydrates such as sugars, sugar derivatives such as alditols, aldonic acids, esterified sugars, and / or sugar polymers may also be used. Some carbohydrate additives of interest include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; lactose, sucrose, trehalose, etc. disaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, and the like. Inorganic and organic salts may include, but are not limited to, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, monobasic sodium phosphate, dibasic sodium phosphate, and any combination thereof.

[0070] In certain embodiments, compositions of the present disclosure may also include antimicrobial agents to prevent or inhibit the growth of microorganisms, such as, for example, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thiomersal, and any combination thereof.

[0071] The composition may also include one or more antioxidants, which reduce or prevent oxidation and therefore deterioration of the composition, and may include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and any combination thereof.

[0072] The compositions of the present disclosure may include one or more surfactants. For example, suitable surfactants may include, but are not limited to, polysorbates such as "Tween 20" and "Tween 80," and pleurolics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as lecithin and other phosphatidylcholines, phospholipids such as phosphatidylethanolamines, fatty acids and fatty acid esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other cations. Acids or bases can also be present in the compositions of the present disclosure. For example, acids can include, but are not limited to, hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, sulfuric acid, fumaric acid, and any combination thereof. Examples of bases include, but are not limited to, sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and any combination thereof.

[0073] The amount of any individual additive in a composition will vary depending on the nature and function of the additive, the dose delivery vehicle, and the particular needs of the composition. In some instances, the optimal amount of an individual additive is determined by routine experimentation, i.e., testing the stability and other parameters of compositions containing various amounts of the additive (ranging from low to high), and then determining the range within which optimal performance is obtained without significant adverse effects. Generally, however, the additive will be present in the composition in an amount of 1% to 99% by weight, such as 5% to 98% by weight, such as 15% to 95% by weight of the additive, including 30% or less by weight, or including 20% ​​or less by weight, or including 10% or less by weight. Pharmaceutical additives that can be used in compositions with additives are listed in "Remington: The Science & Practice." ce of Pharmacy”, 22nd ed., Williams & Willia ms,(2012),the “Physician's Desk Reference ”, 70th ed., PDR Network, Montvale, NJ (2015), and Rowe, R.C., Handbook of Pharmaceutical Exc. ipients,7th ed., Pharmaceutical Press, New New York, NY (2012), each of which is incorporated herein by reference. and is incorporated herein by reference.

[0074] [Usage] Aspects of the present disclosure also include methods of using the compositions (e.g., liquid and lyophilized) described herein. In certain embodiments, the method is a method of deaminating asparagine in a subject. As noted above, the asparaginase enzyme mediates the deamination reaction in which the amino acid asparagine is hydrolyzed to produce aspartic acid and ammonia, for example, according to the following reaction: [ka]

[0075] In some cases, the activity of asparaginase reduces asparagine in a subject, such as reducing the plasma concentration of asparagine in the subject. Depletion of asparagine in a subject adversely affects cells in the subject that depend on the presence of asparagine for protein synthesis. For example, protein synthesis in cells that lack the ability to synthesize asparagine on their own (e.g., cells lacking the enzyme asparagine synthase) can be adversely affected by the lack of exogenous asparagine, resulting in cellular apoptosis. In some examples, cells in a subject that depend on asparagine for protein synthesis may be associated with a neoplastic condition, such as cancer. Accordingly, methods of the present disclosure include methods for treating a neoplastic condition in a subject, such as treating cancer in a subject. Thus, compositions of the present disclosure comprising polyalkylene oxide-asparaginase can be therapeutically effective for treating a neoplastic condition, such as cancer. In certain embodiments, non-neoplastic cells in a subject are not significantly affected by the polyalkylene oxide-asparaginase compositions of the present disclosure. For example, non-neoplastic cells in a subject can possess the enzyme asparagine synthase and thus retain the ability to synthesize asparagine.

[0076] In certain embodiments, the neoplastic condition to be treated in the subject is a polyalkylene oxide- Conditions suitable for treatment by administering asparaginase to a subject include, for example, oncologic conditions that depend on exogenous asparagine. For example, oncologic conditions treatable by administering polyalkylene oxide-asparaginase to a subject include cancers, such as solid tumors or liquid tumors.

[0077] In certain cases, the neoplastic condition is characterized by the presence of a solid tumor. Thus, in some embodiments, the disclosed method is a method of treating a solid tumor in a subject using a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the disclosed method). The method of treating a neoplastic condition in a subject is useful for treating a wide variety of solid tumors, including epithelial and non-epithelial malignancies. Types of solid tumors include, but are not limited to, pancreatic cancer, melanoma, squamous cell carcinoma, non-small cell lung cancer (NSCLC), colon cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, etc. For example, epithelial malignancies treatable using the subject methods include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchial carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell lung cancer and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteosarcoma, epithelial carcinoma, and nasopharyngeal carcinoma, among others.

[0078] Non-epithelial malignancies treatable using the subject method include, but are not limited to, fibrosarcoma, Myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, (osteogenic s These include angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's sarcoma, smooth muscle, rhabdomyosarcoma, and other soft tissue sarcomas.

[0079] Other solid tumors treatable using the subject methods include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0080] In certain cases, the neoplastic condition is characterized by the presence of a liquid tumor. For example, the liquid tumor may include metastatic cancer cells (e.g., circulating tumor cells (CTCs)), blood cancers, and the like, and combinations thereof. Examples of liquid tumors may include, but are not limited to, leukemia, lymphoma, and myeloma. In some examples, the cancer is leukemia. Thus, in some embodiments, the method is a method of treating leukemia in a subject using polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure).

[0081] Various types of leukemia may be suitable for treatment using the subject methods. For example, the leukemia may be acute leukemia. Acute leukemia may be characterized by a rapid increase in immature blood cells. The rapid increase in immature blood cells leads to overcrowding, which in turn causes the bone marrow to produce significantly fewer healthy blood cells. Thus, methods of the present disclosure include, for example, treating acute leukemia in a subject by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) sufficient to treat acute leukemia in the subject.

[0082] In another example, when the leukemia is chronic leukemia, the chronic leukemia is characterized by an increase in relatively mature but abnormal white blood cells. Chronic leukemia takes a longer period (e.g., several months or years) to progress, and abnormal white blood cells are produced at a significantly higher rate than normal. Thus, methods of the present disclosure include methods of treating chronic leukemia in a subject, for example, by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the disclosed composition) effective to treat chronic leukemia in the subject.

[0083] In certain embodiments, the leukemia is lymphoblastic leukemia (also called lymphocytic leukemia). Lymphoblastic leukemia is characterized by the type of blood cell affected by the leukemia. In lymphoblastic leukemia, abnormal changes in blood cells occur in bone marrow cells that normally develop into lymphocytes. For example, lymphoblastic leukemia can be B-cell leukemia. Thus, the methods of the present disclosure include methods of treating lymphoblastic leukemia (lymphoid leukemia) in a subject, for example, by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of the disclosed composition) effective to treat the subject's lymphoblastic leukemia (lymphoid leukemia). For example, a specific type of lymphoblastic leukemia treatable by the subject method includes acute lymphoblastic leukemia (ALL). In these embodiments, the methods of the present disclosure include methods of treating acute lymphoblastic leukemia (ALL) in a subject, for example, by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of a composition of the present disclosure) in an amount effective to treat the subject's ALL. Other types of lymphoblastic leukemia treatable using the subject methods include, but are not limited to, chronic lymphoblastic leukemia (CLLE). These embodiments include methods of treating chronic lymphocytic leukemia (CLL) in a subject, for example, by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of a composition of the present disclosure) effective to treat the subject's chronic lymphocytic leukemia.

[0084] In another embodiment, the leukemia is myeloid leukemia. (also called myelogenous leukemia). Myeloid leukemia can be characterized by blood cells affected by leukemia. In myeloid leukemia, abnormal changes in blood cells occur in bone marrow cells that normally develop into red blood cells and / or platelets. Thus, methods of the present disclosure include, for example, treating myelogenous leukemia in a subject by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of a composition of the present disclosure) effective to treat the subject's myeloid leukemia. For example, a particular type of lymphoblastic leukemia treatable using the subject method is, but is not limited to, acute myeloid leukemia (AML). In these embodiments, the methods of the disclosure include methods of treating acute myeloid leukemia (AML) in a subject, for example, by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase of a composition of the disclosure) effective to treat AML in the subject.

[0085] Examples of AML include, but are not limited to, AML with recurrent cytogenetic translocations, AML with multilineage dysplasia, and other AML. For example, AML with recurrent cytogenetic translocations includes, in particular, AML with t(8;2l)(q22;q22), AMLI (CBF- α) / ETO, acute promyelocytic leukemia (t(l5;l7)(q22;qll-12) and AML with variants, PML / RAR-α), AML with abnormal bone marrow eosinophils (in Includes AML with v(l6)(pl3q22) or t(l6;16)(p13;ql1), CBFb / MYHlIX), and lq23(MLL) abnormalities. Examples of AML with multilineage dysplasia can include those with or without a previous myelodysplastic syndrome. Other types of acute myeloid leukemia include, for example, minimally differentiated AML, undifferentiated AML, differentiated AML, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with acute myelofibrosis.

[0086] Other types of myeloid leukemia treatable using the subject methods include, but are not limited to, chronic myelogenous leukemia (CML). In these embodiments, the methods of the present disclosure include methods for treating chronic myeloid leukemia (CML) in a subject, e.g., by administering to the subject a dose of polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) effective to treat CML in the subject.

[0087] In certain cases, asparaginase can mediate the deamination of glutamine, for example, the amino acid glutamine is hydrolyzed to produce glutamic acid and ammonia according to the following reaction: [ka]

[0088] In some cases, asparaginase activity reduces the concentration of glutamine in a subject, such as reducing plasma glutamine concentrations in the subject. Similar to the discussion above, depletion of glutamine in a subject can adversely affect cells in the subject that depend on the presence of glutamine for protein synthesis. For example, protein synthesis in cells that lack the ability to synthesize glutamine on their own (e.g., cells lacking the enzyme glutamine synthase) can be adversely affected by the lack of exogenous glutamine, resulting in cellular apoptosis. In some examples, cells in a subject that depend on glutamine for protein synthesis may be associated with a neoplastic condition, such as cancer. Accordingly, methods of the present disclosure include methods of treating a neoplastic condition in a subject, such as treating cancer in a subject. Accordingly, methods of the present disclosure include methods of treating a neoplastic condition in a patient, such as treating cancer in a patient, where the neoplastic condition may be dependent on exogenous glutamine. For example, an embodiment of the subject method includes a method of deaminating glutamine in a subject. In certain embodiments, non-neoplastic cells in a subject are not significantly affected by the polyalkylene oxide-asparaginase composition administered to the subject, e.g., non-neoplastic cells in a subject can possess the enzyme glutamine synthase and thus retain the ability to synthesize glutamine.

[0089] As described above, compositions of the present disclosure include lyophilized storage-stable compositions. Prior to administering the composition to a subject, the lyophilized composition is mixed with a liquid to provide a liquid composition suitable for administration, such as by injection or intravenous administration. In some cases, prior to administering the composition to a subject, the lyophilized composition can be combined with water (e.g., water for injection, WFI), e.g., as described above, to provide a liquid composition suitable for injection or intravenous administration. For example, methods of the present disclosure can include reconstituting a lyophilized composition (e.g., a lyophilized storage-stable composition) of the present disclosure. Reconstituting the lyophilized composition can produce a reconstituted dosage unit. In some cases, the reconstituted dosage unit is suitable for administration to a subject, such as by injection or intravenous administration. In certain embodiments, reconstituting a lyophilized composition includes combining the lyophilized composition (e.g., a lyophilized storage-stable composition) with water (e.g., water for injection, WFI).

[0090] The liquid or reconstituted dosage unit contains a predetermined amount of a composition of the present disclosure calculated to produce a desired therapeutic effect in a subject. The amount of composition in a dosage unit (e.g., liquid or reconstituted) administered to a subject can depend on the subject being treated, the severity of the affliction, and the mode of administration. For example, a dosage unit can contain a therapeutically effective amount of a composition as disclosed herein.

[0091] In certain embodiments of the dosage unit, the amount of polyalkylene oxide-asparaginase can include an amount ranging from 100 to 5,000 IU / mL, such as 500 to 4,500 IU / mL, or 500 to 4,000 IU / mL, or 500 to 3,500 IU / mL, or 500 to 3,000 IU / mL, or 500 to 2,500 IU / mL, or 500 to 2,000 IU / mL, or 500 to 1,500 IU / mL, or 500 to 1,000 IU / mL, or 600 to 900 IU / mL, or 700 to 800 IU / mL. In a specific example, the dosage unit may contain an amount in the range of 700 to 800 IU / mL of polyalkylene oxide-asparaginase. For example, the dosage unit may contain 750 IU / mL of polyalkylene oxide-asparaginase.

[0092] In certain embodiments, the dosage unit is 50 IU / mg protein or more, such as 55 IU / mg protein or more, or 60 IU / mg protein or more, or 65 IU / mg g protein or more, or 70 IU / mg protein or more, or 75 IU / mg protein or more, or 80 IU / mg protein or more, or 85 IU / mg protein or more, or 90 IU / mg protein or more, or 95 IU / mg protein or more, or 100 IU / mg protein or more, or 105 IU / mg protein or more, or 110 IU / mg protein or more, or 115 IU / mg protein or more, or 120 IU / mg protein or more, or 125 IU / mg protein or more, or 130 IU / mg protein or more, or 135 IU / mg protein or more, or 140 IU / mg protein or more, or 145 IU / mg protein or more, or 150 IU / mg protein or more.

[0093] For example, the dosage unit may have a specific activity of 85 IU / mg protein or greater. In some embodiments, the dosage unit has a specific activity in the range of 50-150 IU / mg protein, or 55-145 IU / mg protein, or 60-140 IU / mg protein, or 65-135 IU / mg protein, or 70-130 IU / mg protein, or 75-125 IU / mg protein, or 80-120 IU / mg protein, or 85-115 IU / mg protein, or 90-110 IU / mg protein, or 95-105 IU / mg protein. In some cases, the dosage unit has a specific activity in the range of 65-140 IU / mg protein, or 70-135 IU / mg protein, or 75-130 IU / mg protein, or 75-125 IU / mg protein, etc. For example, the dosage unit may have a specific activity in the range of 75 to 125 IU / mg protein.

[0094] In certain embodiments, the dosage unit has a concentration of 1 mg / mL to 15 mg / mL, 1.5 mg / mL to 14.5 mg / mL, etc., or 2 mg / mL to 14 mg / mL, or 2.5 mg / mL to 13.5 mg / mL, or 3 mg / mL to 13 mg / mL, or 3.5 mg / mL to 12.5 mg / mL, or 4 mg / mL to 12 mg / mL, or 4.5 mg / mL to 11.5 mg / mL, or 4.5 mg / mL to 11 mg / mL. In some cases, the dosage unit contains a therapeutically effective amount (e.g., protein concentration) of polyalkylene oxide-asparaginase in the range of 1 mg / mL to 15 mg / mL, such as 4.5 mg / mL to 10.5 mg / mL, or 4.5 mg / mL to 10 mg / mL, or 4.5 mg / mL to 9.5 mg / mL, or 4.5 mg / mL to 9 mg / mL, or 4.5 mg / mL to 8.5 mg / mL, or 5 mg / mL to 8 mg / mL.

[0095] When administered to a subject, the dosage unit is 500-5,000 IU / m 2or 500-4,500 IU / m2, or 500-4,000 IU / m2, or 500-3,500 IU / m2, or 500-3,000 IU / m2, or 1,000-3,000 IU / m2, or 1,500-3,000 IU / m2, or 1,750-3,000 IU / m2, or 2,000-3,000 IU / m2, or 2,000-2,750 IU / m2, or 2,250-2,750 IU / m2. For example, the dosage unit may deliver 1,000 to 3,000 IU / m2 of polyalkylene oxide-asparaginase to a subject. In certain examples, the dosage unit may deliver 2,000 to 2,750 IU / m2 of polyalkylene oxide-asparaginase to a subject. In certain examples, the dosage unit may deliver 2,250 to 2,750 IU / m2 of polyalkylene oxide-asparaginase to a subject. For example, the dosage unit may deliver 2,500 IU / m2 of polyalkylene oxide-asparaginase to a subject.

[0096] In certain embodiments, the dosage unit may include a buffer such as those described above, for example, the dosage unit may include a phosphate buffer, and as such, may include sodium phosphate dibasic and sodium phosphate monobasic.

[0097] In some cases, the dosage unit includes a phosphate buffer solution, and as such, includes dibasic sodium phosphate and monobasic sodium phosphate. In certain examples, the dosage unit includes dibasic sodium phosphate in an amount ranging from 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 2 to 7 mg / g, or 3 to 6 mg / g, or 4 to 6 mg / g, or 5 to 6 mg / g. For example, the dosage unit may include dibasic sodium phosphate in an amount ranging from 4 to 6 mg / g. In certain examples, the dosage unit may include dibasic sodium phosphate in an amount ranging from 5 to 6 mg / g. For example, the dosage unit may include dibasic sodium phosphate in an amount of about 5.5 mg / g, such as 5.6 mg / g (or 5.58 mg / g). In certain embodiments, the dosage unit contains an amount of monobasic sodium phosphate in the range of 0.05 to 5 mg / g, such as 0.1 to 4.5 mg / g, or 0.1 to 4 mg / g, or 0.1 to 3.5 mg / g, or 0.1 to 3 mg / g, or 0.1 to 2.5 mg / g, or 0.1 to 2 mg / g, or 0.5 to 2 mg / g, or 1 to 2 mg / g, or 1 to 1.5 mg / g. For example, the dosage unit may contain an amount of monobasic sodium phosphate in the range of 1 to 2 mg / g. In certain examples, the dosage unit may contain an amount of monobasic sodium phosphate in the range of 1 to 1.5 mg / g. For example, the dosage unit may contain an amount of monobasic sodium phosphate in the range of 1.2 mg / g (or 1.29 mg / g).

[0098] In certain embodiments, the dosage unit includes a salt. Salts suitable for use in the dosage unit include salts that are compatible with polyalkylene oxide-asparaginase and are suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., and combinations thereof. In certain examples, the dosage unit includes a salt such as sodium chloride. In some cases, the dosage unit contains salt (e.g., sodium chloride) in an amount in the range of 1 to 20 mg / g, such as 1 to 19 mg / g, or 1 to 18 mg / g, or 1 to 17 mg / g, or 1 to 16 mg / g, or 1 to 15 mg / g, or 2 to 15 mg / g, or 3 to 15 mg / g, or 4 to 15 mg / g, or 5 to 14 mg / g, or 5 to 13 mg / g, or 5 to 12 mg / g, or 5 to 11 mg / g, or 5 to 10 mg / g, or 5 to 9 mg / g, or 6 to 9 mg / g, or 7 to 9 mg / g, or 8 to 9 mg / g.

[0099] For example, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 1-10 mg / g. In particular examples, the dosage unit may contain an amount of salt (e.g., sodium chloride) in the range of 5-10 mg / g. In particular examples, the dosage unit contains an amount of salt (e.g., sodium chloride) in the range of 6-9 mg / g. In particular examples, the dosage unit contains an amount of salt (e.g., sodium chloride) in the range of 8-9 mg / g. For example, the dosage unit contains salt (e.g., sodium chloride) in an amount of 8.5 mg / g.

[0100] In one example, the formulation administered to the subject is a PEG-asparagine formulation commercially known as Oncaspar®, which has been approved for commercial marketing by the U.S. Food and Drug Administration. Oncaspar® (pegaspargase) is a liquid injectable formulation of L-asparaginase (L-asparagine amidohydrolase) covalently linked to monomethoxypolyethylene glycol (mPEG) in a clear, colorless, preservative-free solution in isotonic, sterile phosphate-buffered saline, pH 7.3. Each milliliter contains 750±150 international units of sodium phosphate, dibasic, USP. (5.58 mg), Sodium Phosphate, Monobasic, USP (1.20 mg) and Sodium Chloride, USP (9.5 mg), in Water for Injection, USP.

[0101] In certain embodiments, the dosage unit administered to a subject is a dosage unit prepared from a lyophilized composition, such as a storage-stable lyophilized composition described herein. The dosage unit prepared from a lyophilized composition may be a liquid formulation. In addition to polyalkylene oxide-asparaginase, embodiments of the dosage unit (e.g., a dose reconstituted from a lyophilized composition) may include a buffer, salt, and sugar.

[0102] In certain embodiments, the reconstituted dosage unit includes a buffer, such as those described in detail above. In some cases, the reconstituted dosage unit includes dibasic sodium phosphate in an amount ranging from 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 1 to 6 mg / g, or 1 to 5 mg / g, or 1 to 4 mg / g, or 2 to 4 mg / g, or 2 to 3 mg / g, or 2.5 to 3 mg / g. For example, the reconstituted dosage unit may include dibasic sodium phosphate in an amount ranging from 1 to 5 mg / g. In certain instances, the reconstituted dosage unit may include dibasic sodium phosphate in an amount ranging from 2 to 4 mg / g. In certain instances, the reconstituted dosage unit may include dibasic sodium phosphate in an amount ranging from 2.5 to 3 mg / g. For example, the reconstituted dosage unit may contain about 3 mg / g, such as 2.8 mg / g (or 2.79 mg / g), of dibasic sodium phosphate. In certain embodiments, the reconstituted dosage unit contains monobasic sodium phosphate in an amount ranging from 0.05 to 1 mg / g, such as 0.1 to 0.9 mg / g, or 0.1 to 0.8 mg / g, or 0.2 to 0.8 mg / g, or 0.3 to 0.8 mg / g, or 0.4 to 0.8 mg / g, or 0.45 to 0.75 mg / g, or 0.5 to 0.7 mg / g. For example, the reconstituted dosage unit may contain monobasic sodium phosphate in an amount ranging from 0.45 to 0.75 mg / g. In certain examples, the reconstituted dosage unit contains monobasic sodium phosphate in an amount ranging from 0.5 to 0.7 mg / g. For example, the reconstituted dosage unit contains monobasic sodium phosphate in an amount of 0.6 mg / g.

[0103] In certain embodiments, the reconstituted dosage unit includes a salt, such as those salts described in detail above. In certain examples, the reconstituted dosage unit includes a salt, such as sodium chloride. In some cases, the reconstituted dosage unit includes a salt (e.g., sodium chloride) in an amount ranging from 0.5 to 10 mg / g, such as 1 to 9 mg / g, or 1 to 8 mg / g, or 1 to 7 mg / g, or 1 to 6 mg / g, or 1 to 5 mg / g, or 2 to 5 mg / g, or 3 to 5 mg / g, or 4 to 5 mg / g, or 4 to 4.5 mg / g. For example, the reconstituted dosage unit includes a salt (e.g., sodium chloride) in an amount ranging from 1 to 10 mg / g. In certain examples, the reconstituted dosage unit includes a salt (e.g., sodium chloride) in an amount ranging from 3 to 5 mg / g. In certain examples, the reconstituted dosage unit includes a salt (e.g., sodium chloride) in an amount ranging from 4 to 4.55 mg / g. For example, the reconstituted dosage unit contains about 4 mg / g of salt (eg, sodium chloride), such as 4.25 mg / g.

[0104] In certain embodiments, the reconstituted dosage unit includes a sugar, such as a sugar described in detail above. In certain examples, the reconstituted dosage unit includes a sugar, such as a disaccharide. In some cases, the reconstituted dosage unit includes a sugar, such as sucrose. In some cases, the reconstituted dosage unit includes a sugar (e.g., sucrose) in an amount ranging from 1 to 250 mg / g, such as 5 to 200 mg / g, or 10 to 150 mg / g, or 10 to 100 mg / g, or 10 to 90 mg / g, or 10 to 80 mg / g, or 20 to 70 mg / g, or 20 to 60 mg / g, or 30 to 50 mg / g, or 40 to 50 mg / g. For example, the reconstituted dosage unit includes an amount of sugar (e.g., sucrose) in the range of 10 to 100 mg / g. In certain examples, the reconstituted dosage unit includes a sugar (e.g., sucrose) in the range of 30 to 50 mg / g. In particular examples, the reconstituted dosage unit may contain an amount of sugar (e.g., sucrose) in the range of 40-50 mg / g. For example, the reconstituted dosage unit may contain an amount of sugar (e.g., sucrose) of 45 mg / g.

[0105] In certain embodiments, the dosage unit (e.g., liquid or reconstituted) has a pH range compatible with physiological conditions. In some cases, the pH of the dosage unit is in the range of 7 to 8. For example, the pH of the dosage unit may be in the range of 7 to 7.5. In some cases, the pH of the dosage unit is 7.2. In some cases, the pH of the dosage unit is 7.3. In some cases, the pH of the dosage unit is 7.4.

[0106] In certain embodiments, the method can include administering the dosage unit to a subject to deaminate asparagine in the subject. The route of administration can be selected according to various factors, including, but not limited to, the condition to be treated, the type of composition and / or device used, and the subject to be treated. Routes of administration useful in the disclosed methods include, but are not limited to, oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), intramuscular (im), rectal, topical, intraocular, nasal, and transdermal. For example, compositions suitable for injection can be administered intravenously, intramuscularly, intradermally, subcutaneously, sublingually, intraosseously, or by other routes of administration. In some instances, administering the dosage unit to the subject comprises administering the dosage unit intravenously to the subject. In some instances, administering the dosage unit to the subject comprises administering the dosage unit intramuscularly to the subject.

[0107] In some instances, administering the reconstituted dosage unit to a subject comprises administering the reconstituted dosage unit intravenously to a subject, hi some instances, administering the reconstituted dosage unit to a subject comprises administering the reconstituted dosage unit intramuscularly to a subject.

[0108] In certain embodiments, the method includes administering the dosage unit according to a therapeutic regimen. For example, in some cases, the subject to be treated can be prescribed a therapeutic regimen by a healthcare provider. In some cases, the therapeutic regimen includes, but is not limited to, five doses per day, four doses per day, three doses per day, two doses per day, once per day, three doses per week, twice per week, once per week, once per two weeks, once per three weeks, once per month, once per five weeks, once per six weeks, once per seven weeks, once every two months, and any combination thereof.

[0109] In some embodiments, the treatment regimen involves administering one or more doses over an extended period of time. In certain cases, a single dose (e.g., a single dosage unit) can be administered to a subject, and following the initial administration, one or more doses can be administered to the subject over a subsequent period of time. In certain instances, one or more doses (e.g., one or more dosage units) can be administered to a subject, and following the initial administration, one or more doses can be administered to the subject over a subsequent period of time. For example, a single dose (e.g., a single dosage unit) can be administered to a subject, and following this single administration, single doses can be administered to the subject over a subsequent period of time. In some cases, one or more doses (e.g., For example, a single dose (e.g., one or more dosage units) can be administered to a subject, and following an initial dose, one or more doses can be administered to a subject at subsequent time points. In some cases, a single dose (e.g., a single dosage unit) can be administered to a subject, and following this single dose, a single dose can be administered to the subject at a subsequent time point. Additional single doses can be administered to the subject at subsequent time points. In other cases, a single dose (e.g., a single dosage unit) can be administered to a subject, and following this single dose, two or more doses can be administered at subsequent time points. Additional single or multiple doses can be administered at subsequent time points. Additional single doses can be administered to a subject at subsequent time points.

[0110] In other cases, a single dose (e.g., a single dosage unit) can be administered to a subject, and Following this single administration, two or more doses can be administered at subsequent time points. In certain examples, the therapeutic regimen includes multiple phases. The therapeutic regimen includes multiple phases, with the administration schedule being different for each phase of the therapeutic regimen. In some cases, a subject is prescribed a therapeutic regimen having two phases, an induction phase and a consolidation phase. In certain examples, a subject is prescribed a therapeutic regimen having two phases, with the administration schedule of the first phase being different from the administration schedule of the second phase. For example, a subject is prescribed a therapeutic regimen having two phases, an induction phase and a consolidation phase, with the administration schedule of the induction phase being different from the administration schedule of the consolidation phase. In other embodiments, a subject is prescribed a therapeutic regimen having three phases, including an induction phase, a consolidation phase, and a maintenance phase. In some examples, a subject is prescribed a therapeutic regimen having three phases, an induction phase, a consolidation phase, and a maintenance phase, with the administration schedule of each phase being different from the other phases. For example, a subject may be prescribed a treatment regimen that includes three phases: an induction phase, a consolidation phase, and a maintenance phase, where the dosing schedule for each phase is different from each of the other phases.

[0111] In certain embodiments, the length of treatment time for each phase of the treatment regimen can be the same or, in other cases, different. For example, the length of time between the induction phases can be one week or more, such as two weeks or more, or three weeks or more, or four weeks or more, or five weeks or more, or six weeks or more, or seven weeks or more, or eight weeks or more. In some cases, the length of time between the induction phases is four weeks. In some cases, the length of time between the induction phases is four weeks. It is five weeks long.

[0112] In certain embodiments, the length of time between coupling phases is 1 week or more, such as 2 weeks or more, or 3 weeks or more, or 4 weeks or more, or 5 weeks or more, or 6 weeks or more, or 7 weeks or more, or 8 weeks or more, or 9 weeks or more, or 10 weeks or more, or 11 weeks or more, or 12 weeks or more, or 13 weeks or more, or 14 weeks or more, or 15 weeks or more, or 16 weeks or more, or 17 weeks or more, or 18 weeks or more, or 19 weeks or more, or 20 weeks or more, or 21 weeks or more, or 22 weeks or more, or 23 weeks or more, or 24 weeks or more, or 25 weeks or more, or 26 weeks or more, or 27 weeks or more, or 28 weeks or more, or 29 weeks or more, or 30 weeks or more, or 31 weeks or more, or 32 weeks or more. In some embodiments, the length of time between coupling phases is 8 weeks. In some embodiments, the length of time between coupling phases is 27 weeks. In some embodiments, the length of time between coupling phases is 30 weeks.

[0113] In certain embodiments, the length of time during the maintenance phase is 2 weeks or more, or 3 weeks or more, or 4 weeks or more, or 5 weeks or more, or 6 weeks or more, or 7 weeks or more, or 8 weeks or more, or 9 weeks or more, or 10 weeks or more, or 12 weeks or more, or 16 weeks or more, or 20 weeks or more, or 24 weeks or more, or 28 weeks or more, or 32 weeks or more, or 36 weeks or more, or 40 weeks or more, or 44 weeks or more, or 48 weeks or more, or 52 weeks or more, or 56 weeks or more, or 60 weeks or more, or 64 weeks or more, or 68 weeks or more, or 72 weeks or more, or 76 weeks or more, or 80 weeks or more, or 84 weeks or more, or 88 weeks or more, or 92 weeks or more, or 96 weeks or more, or 100 weeks or more, or 104 weeks or more, or 108 weeks or more, or 112 weeks or more, or 116 weeks or more, or 120 weeks or more, or 124 weeks or more, or 128 weeks or more, or 132 weeks or more, or 136 weeks or more, or 140 weeks or more, or 144 weeks or more, or 148 weeks or more, or 152 weeks or more, or 156 weeks or more, or 160 weeks or more, or 164 weeks or more, or 168 weeks or more, or 172 weeks or more, or 176 weeks or more, or 180 weeks or more, etc. In some cases, the length of time between maintenance phases is 8 weeks. In some cases, the length of time between maintenance phases is 88 weeks. In some cases, the length of time between maintenance phases is 104 weeks. In some cases, the length of time between consolidation phases is 140 weeks. In some cases, the length of time between maintenance phases is 156 weeks. The length of time between maintenance phases ranges from 88 to 104 weeks. In some cases, the length of time between maintenance phases ranges from 88 to 140 weeks. In some cases, the length of time between maintenance phases ranges from 88 to 156 weeks.

[0114] Examples of therapeutic regimens that can be administered to a subject include, but are not limited to, those described herein. In certain embodiments, the therapeutic regimen comprises administering a single dosage unit to the subject during the induction phase and administering multiple dosage units during the maintenance phase. In certain embodiments, the therapeutic regimen comprises administering a single dosage unit to the subject during the induction phase and administering multiple dosage units during the consolidation phase. For example, the multiple dosage units can be administered by administering a dosage unit to the subject every three weeks (e.g., during the consolidation phase). In some cases, a single dosage unit can be administered to the subject once every three weeks. As described above, the consolidation phase can be 30 weeks long, and therefore a total of 10 dosage units can be administered to the subject (e.g., a single dosage unit can be administered to the subject once every three weeks for 30 weeks). Additional (or fewer) dosage units can be administered to the subject during or after the induction and consolidation phases, as described or as prescribed by a healthcare provider.

[0115] In other examples, the treatment regimen can involve administering a single dosage unit to the subject during the induction phase and multiple dosage units during the consolidation phase, where multiple dosage units can be administered to the subject by administering a dosage unit to the subject every two weeks. In some cases, a single dosage unit is administered to the subject every two weeks. As noted above, the consolidation phase can be 30 weeks long, and thus a total of 15 dosage units can be administered to the subject (e.g., a single dosage unit can be administered to the subject once every two weeks for 30 weeks). Additional (or fewer) dosage units can be administered to the subject during or after the induction and consolidation phases, as described or prescribed by a healthcare provider.

[0116] In other embodiments, the therapeutic regimen includes administering a single dosage unit to the subject during the induction phase, multiple dosage units during the consolidation phase, and multiple dosage units during the maintenance phase. For example, multiple dosage units during the consolidation phase can be administered to the subject on a specific day following the initiation of the consolidation phase. In some instances, the multiple dosage units during the consolidation phase can be administered to the subject by simultaneously administering two or more dosage units to the subject. For example, the multiple dosage units during the consolidation phase can be administered by administering two or more dosage units to the subject on a specific day following the initiation of the consolidation phase, and two or more dosage units can be administered on subsequent days during the consolidation phase. An example of this type of therapeutic regimen can include administering two dosage units to the subject on day 15 following the initiation of the consolidation phase and two dosage units on day 43 following the initiation of the consolidation phase. Additional (or fewer) dosage units can be administered to the subject during the induction phase and consolidation phase, or can be administered after the consolidation phase but before the maintenance phase, if desired or prescribed by a healthcare provider.

[0117] In certain embodiments, multiple dosage units during the maintenance phase can be administered to a subject on a specific day following the initiation of the maintenance phase. In some examples, multiple dosage units can be administered to a subject during the maintenance phase by simultaneously administering two or more dosage units to the subject. For example, multiple dosage units during the maintenance phase can be administered to a subject on a specific day following the initiation of the maintenance phase, and two or more dosage units can be administered on subsequent days during the maintenance phase. An example of this type of treatment regimen can include administering two dosage units to a subject on day 2 following the initiation of the maintenance phase, and administering two dosage units on day 22 following the initiation of the maintenance phase. Another example of a treatment regimen during the maintenance phase can include administering two dosage units to a subject on day 4 following the initiation of the maintenance phase, and administering two dosage units on day 43 following the initiation of the maintenance phase. In some cases, a treatment regimen can include multiple maintenance phases. In certain examples, the dosing schedule during each maintenance phase can be the same, or in other examples, the dosing schedule during each maintenance phase can be different. If desired or prescribed by a healthcare provider, additional (or fewer) dosage units can be administered to the subject during the consolidation and maintenance phases, or after the maintenance phase, or during a different maintenance phase.

[0118] In certain embodiments, the dosage units of the present disclosure can be administered prior to, simultaneously with, or subsequent to other active agents for the treatment of related or unrelated conditions, such as in combination therapy. Examples of such additional therapies include radiation therapy, surgical therapy, and chemotherapy. When provided simultaneously with other active agents, the dosage units of the present disclosure can be provided in the same or different formulations. For example, combination therapy can be achieved by administering a pharmaceutical composition having a dosage unit and another active agent, such as at least one chemotherapeutic agent, that in combination provides a therapeutically effective dose according to a specific treatment regimen. The administration of the separate pharmaceutical compositions can be simultaneous or at different times (e.g., sequentially, on the same day, or on different days, in either order), so long as the combination of these agents causes a therapeutically effective effect in the subject being treated.

[0119] Accordingly, aspects of the present disclosure further include combination therapy. In certain embodiments, the subject method includes administering a therapeutically effective amount of one or more additional active agents. Combination therapy means that a polyalkylene oxide-asparaginase (e.g., as described herein) can be used in combination with other therapeutic agents for treating other individual diseases or conditions. In certain embodiments, a compound of the present disclosure is administered simultaneously with the administration of the other therapeutic agent, either as a component of a composition comprising a compound of the present disclosure or as a component of a different composition. In certain embodiments, a composition comprising a compound of the present disclosure is administered prior to or subsequent to the administration of the other therapeutic agent.

[0120] The subject compounds can be used in conjunction with any agent useful in treating neoplastic conditions, such as anti-cancer and anti-tumor agents. One class of anti-cancer agents of interest is chemotherapeutic agents. "Chemotherapy" refers to the administration of one or more chemotherapeutic and / or other agents to a cancer patient by a variety of methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, or inhalation. Agents of interest that can be used in conjunction with the subject compounds include, but are not limited to, cancer chemotherapeutic agents, agents that act to reduce cell proliferation, antimetabolite agents, microtubule-active agents, hormone modulators, and steroids, natural products, and biological response modifiers, e.g., as described in more detail below.

[0121] Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells, and encompass cytotoxic and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds may also be used. Suitable cancer chemotherapeutic agents include dolastatin and its active analogs and derivatives; and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, e.g., WO 96 / 33212, WO 96 / 14856, and U.S. Patent No. 6,323,315. For example, dolastatin 10 or auristatin PE can be included in the antibody-drug conjugates of the present disclosure. Suitable Suitable cancer chemotherapeutic agents include maytansinoids and their active analogs and derivatives (e.g., , EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); Zuokarmay KW-sin and its active analogs and derivatives (e.g., its synthetic analogs, KW- 2189 and CB1-TMl); and benzodiazepines and their active analogs Also included are analogs and derivatives (e.g., pyrrolobenzodiazepines (PBDs)).

[0122] Drugs that act to reduce cell proliferation are well known and widely used in the art. Such drugs include nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazines, and include, but are not limited to, mechlorethamine, cyclophosphamide (Cytoxan), and cyclosporin (Cyclosporin). TM ), melphalan (L-sarcolysin), carmustine (BCNU), lomustine ( CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, These include alkylating agents including uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0123] Antimetabolite agents include, but are not limited to, cytarabine (CYTOSAR-U), cytosine arabine (CYTOSAR-U), Rabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, methotrexate, 10- Propargyl-5,8-dideazatifolate (PDDF, CB3717), 5,8-dideazate Folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including dihydrofolic acid (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0124] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include, but are not limited to, ara- C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, azathioprine; brequina brequinar; alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, such as etoposide, teniposide, etc.; antibiotics, such as anthracyclines, daunorubicin hydrochloride (daunomycin, rubidomycin, cerubidine™, idarubicin, doxorubicin, epirubicin, and morpholino derivatives, etc.); phenoxizone biscyclopeptides, such as dactinomycin; basic groups Glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mithramycin); anthracenediones, e.g., mitoxantrone; azirinopyrroindolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, prograf, rapamycin, etc.).

[0125] Other antiproliferative cytotoxic agents include navelbine, CPT-11, and ana These are anastrazole, letrozole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine. Microtubule-active agents having antiproliferative activity and suitable for use include, but are not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysteine, vinblastine sulfate, vincristine sulfate, and, but not limited to, epothilone A, epothilone B, and discodermolide. Natural and synthetic epothilones; including estramustine, nocodazole, etc.

[0126] Hormone modulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, corticosteroids, e.g., prednisone, dextromethorphan, benzodiazepines, benzocaine ... methasone, etc.; estrogens and progestins, such as hydroxyprogesterone caprylate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, etc.; and adrenocortical suppressants, such as aminoglutethimide; 17a-ethinylestradiol; diethylstilbestrol, testosterone, fluoxetine, etc. These include oxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil®), toremifene (Fareston®), and Zoladex®. Estrogens stimulate proliferation and differentiation; therefore, compounds that bind to the estrogen receptor are used to block this activity. Corticosteroids can inhibit T-cell proliferation.

[0127] Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin, platins; ureas, e.g., hydroxyurea; and hydrazines, e.g., N-methylhydrazine Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid, thalidomide, desoxyspergualin, azaserine, leflunomide, mizoribine, azaspirone (SKF105685); Iressa® (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-( 4-morpholinyl)propoxy)quinazoline); and the like.

[0128] Taxanes are suitable for use. "Taxane" includes any active taxane derivative or prodrug, as well as paclitaxel. "Paclitaxel" (as used herein, e.g., docetaxel, TAXOL) TM ,TAXOTERE TM(Formulations of docetaxel, which should be understood to include analogs, formulations, and derivatives such as the 10-desacetyl analog of paclitaxel and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be readily synthesized using techniques well known to those skilled in the art (WO94 / 07882, WO94 / 07881, WO94 / 07880, see also WO94 / 07876, WO93 / 23555, WO93 / 10076; U.S. Patents Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or, for example, Sigma Chemical Co., St. Louis, Missouri (T7 from Pacific yew (Taxus brevifolia) Paclitaxel is available from a variety of commercial sources, including paclitaxel T-402 from Taxus yannanensis; or T-1912 from Taxus yannanensis. Paclitaxel should not be understood to refer only to the commonly chemically available forms of paclitaxel, but also to analogs and derivatives (e.g., the Taxotere compounds listed above). TM The term "taxane" refers to hydrophilic derivatives, as well as hydrophobic derivatives such as paclitaxel, docetaxel, and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application WO 99 / 18113; the piperazino and other derivatives described in WO 99 / 14209; the taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO 98 / 28288; U.S. Patent No. 5,821, 263; and taxol derivatives described in U.S. Patent No. 5,415,869. This further includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Patent No. 5,824,701.

[0129] Suitable biological response modifiers for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) antagonists of tumor-associated antigens, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) coagulants; Ronnie's stimulating factor; and (9) angiogenesis inhibitors.

[0130] Subjects treatable with the disclosed methods and compositions can include subjects of any age. In some cases, the subject can be an adult. For example, an adult human subject is 18 years of age or older. Subjects treatable with the disclosed methods and compositions can include juvenile subjects. For example, a juvenile human subject can be under 18 years of age. In some examples, the subject ranges in age from 1 month to 18 years, such as 1 year to 18 years, including 2 years to 18 years, including 5 years to 16 years, etc.

[0131] In some examples, the method includes diagnosing whether a subject has AML. A subject can be diagnosed with AML using any conventional procedure. In some examples, the French, American, and British (FAB) classification system can be used to diagnose and classify acute myeloid leukemia. A diagnosis of acute myeloid leukemia requires that myeloblasts constitute 30% or more of the bone marrow cells or circulating leukocytes (or 20% based on the latest World Health Organization (WHO) classification system). The hematological nature of the disease defines various subtypes, which are described below. The FAB nomenclature (M1 to M7) classifies acute myeloid leukemia subtypes according to the most closely related normal bone marrow element. In some examples, the method includes determining the suitability of a subject diagnosed with AML for treatment with a polyalkylene oxide asparaginase composition, e.g., as described herein. In some examples, the method includes monitoring the effectiveness of the treatment. The effectiveness of the treatment can be monitored using any conventional procedure.

[0132] [Manufacturing method] Aspects of the present disclosure include methods for producing the polyalkylene oxide-asparaginase compositions described herein. In certain cases, the methods are methods for producing liquid polyalkylene oxide-asparaginase compositions described herein. The methods can include producing an aqueous composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently attached to the asparaginase by a linker, a buffer, and a salt.

[0133] An embodiment of a method for producing a polyalkylene oxide-asparaginase composition can include producing an aqueous concentrated composition. For example, the method for producing the aqueous concentrated composition can include preparing a solution of asparaginase (e.g., L-asparaginase); The method may include one or more of the steps of: attaching a polyethylene oxide (e.g., polyethylene glycol) to the asparaginase; purifying the polyalkylene oxide-asparaginase; filtering and concentrating the solution of polyalkylene oxide-asparaginase; diluting the solution of polyalkylene oxide-asparaginase; filtering the solution of polyalkylene oxide-asparaginase and filling the solution of polyalkylene oxide-asparaginase into sterile containers; and storing the solution of polyalkylene oxide-asparaginase.

[0134] In the method for producing an aqueous concentrate composition, asparaginase (e.g., L-asparaginase) A solution of asparaginase can be prepared. The asparaginase can be mixed with an aqueous solution (e.g., a buffered aqueous solution). Examples of suitable buffers include, but are not limited to, phosphate buffer, phosphate buffered saline (PBS), Dulbecco's phosphate buffered saline (PBS), and phosphate buffered saline (PBS). Examples of suitable buffers include phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), Tris buffer, Ringer's lactate buffer, borate buffer, and the like, and combinations thereof. In some cases, asparaginase is mixed with a phosphate buffer.

[0135] The phosphate buffer can include dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the aqueous concentrate composition is in the range of 0.05 to 5 wt.%, such as 0.1 to 4.5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3.5 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2.5 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1 wt.%, or 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.2 to 0.6 wt.%, or 0.3 to 0.6 wt.%, or 0.4 to 0.6 wt.%, or 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate may be present in the aqueous concentrate composition in an amount ranging from 0.1 to 1 wt.%. In certain examples, dibasic sodium phosphate may be present in the composition in an amount of 0.2 to 0.8 wt.%. In certain examples, dibasic sodium phosphate may be present in the composition in an amount of 0.3 to 0.6 wt.%. In certain examples, dibasic sodium phosphate may be present in the composition in an amount of 0.5 to 0.6 wt.%. For example, dibasic sodium phosphate may be present in the composition in an amount of about 0.6 wt.%, such as 0.56 wt.% (or 0.558 wt.%). In certain embodiments, the amount of monobasic sodium phosphate in the aqueous concentrate composition is 0.01-1.8 wt.%, or 0.01-1.6 wt.%, or 0.01-1.4 wt.%, or 0.01-1.2 wt.%, or 0.01-1.0 wt.%, or 0.01-0.8 wt.%, or 0.01-0.6 wt.%, or 0.01-0.4 wt.%, or 0.01 It can be present in an amount ranging from 0.005 to 2 wt.%, such as 0.005 to 2 wt.%, or 0.02 to 0.18 wt.%, or 0.03 to 0.16 wt.%, or 0.04 to 0.16 wt.%, or 0.045 to 0.15 wt.%, or 0.04 to 0.14 wt.%, or 0.05 to 0.14 wt.%, or 0.1 to 0.2 wt.%, or 0.1 to 0.15 wt.%. For example, monobasic phosphoric acid can be present in the aqueous concentrate composition in an amount ranging from 0.05 to 0.2 wt.%. In a particular example, monobasic sodium phosphate can be present in the composition in an amount of 0.01 to 0.2 wt.%.In certain examples, monobasic sodium phosphate can be present in the composition in an amount of 0.09-0.15 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in an amount of 0.09-0.2 wt.%. In certain examples, monobasic sodium phosphate can be present in the composition in an amount of 0.1-0.15 wt.%. For example, monobasic sodium phosphate can be present in the composition in an amount of 0.12 wt.% (or 0.129 wt.%).

[0136] Additional components that may be included in the aqueous concentrate composition include salts. Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and the like, and combinations thereof. In a particular example, the salt is sodium chloride.

[0137] In some cases, the amount of salt (e.g., sodium chloride) in the composition in the aqueous concentrate is in the range of 0.05 to 5 wt.%, such as 0.05 to 4 wt.%, or 0.05 to 3 wt.%, or 0.05 to 2 wt.%, or 0.1 to 5 wt.%, or 0.1 to 4 wt.%, or 0.1 to 3 wt.%, or 0.1 to 2 wt.%, or 0.1 to 1.5 wt.%, or 0.1 to 1 wt.%, or 0.2 to 1 wt.%, or 0.3 to 1 wt.%, or 0.4 to 1 wt.%, or 0.5 to 1 wt.%, or 0.6 to 1 wt.%, or 0.7 to 1 wt.%, or 0.8 to 1 wt.%, or 0.8 to 0.9 wt.%. For example, the salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.5 to 1 wt.%. For example, the salt (e.g., sodium chloride) can be present in the composition in an amount ranging from 0.2 to 2 wt.%. In a particular example, the salt (e.g., sodium chloride) can be present in an amount ranging from 0.7 to 1 wt.%. In certain instances, the salt (e.g., sodium chloride) may be present in the composition in an amount ranging from 0.8 to 0.9 wt.%. For example, the salt (e.g., sodium chloride) may be present in the composition in an amount of 0.85 wt.%.

[0138] Other aspects of the present disclosure include methods for producing the lyophilized storage-stable compositions described herein. In certain cases, the methods are methods for producing the lyophilized polyalkylene oxide-asparaginase compositions described herein. The methods can include lyophilizing an aqueous composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently attached to the asparaginase by a linker, a buffer, a salt, and a sugar in a manner sufficient to produce the lyophilized storage-stable polyalkylene oxide-asparaginase composition.

[0139] In certain embodiments, freeze-drying is used to dehydrate an aqueous concentrated composition. In some instances, freeze-drying involves removing water from an aqueous concentrated composition. This water can be removed by sublimating the water in the composition. For example, the water in the composition can undergo a phase transition from solid to gas. In certain cases, freeze-drying involves freezing the composition (e.g., freezing the water in the composition) and then reducing the pressure around the composition so that the water in the composition undergoes sublimation. During freeze-drying, the temperature of the composition can be reduced, for example, to a temperature below the freezing point of water in the composition. For example, the temperature in the composition can be 0°C or lower, or -5°C or lower, or -10°C or lower, or -15°C or lower. , or drops to -20°C or below, or -25°C or below, or -30°C or below, or -35°C or below, or -40°C or below, or -45°C or below, or -50°C or below, or -55°C or below, or -60°C or below, or -65°C or below, or -75°C or below In some cases, the temperature in the composition is reduced to -45°C. In this case, the temperature in the composition is reduced to -30°C.

[0140] In certain embodiments, the pressure surrounding the composition is reduced to less than standard atmospheric pressure. For example, the pressure surrounding the composition can be reduced to 500 T or less, such as 250 T or less, or 100 T or less, or 50 T or less, or 10 T or less, or 1 T or less, or 500 mT or less, or 400 mT or less, or 300 mT or less, or 200 mT or less, or 100 mT or less, or 90 mT or less, or 80 mT or less, or 70 mT or less, or 60 mT or less, or 50 mT or less, or 40 mT or less, or 30 mT or less, or 20 mT or less, or 10 mT or less. In some cases, the pressure surrounding the composition is reduced to 60 mT or less, such as 050 mT or less.

[0141] In some embodiments, freeze-drying can include increasing the temperature of the composition while reducing the pressure surrounding the composition. For example, the temperature of the composition can be increased from a minimum temperature as described above to a higher temperature. In some cases, the temperature is increased to promote sublimation of water in the composition at reduced ambient pressure.

[0142] Embodiments of methods for producing a lyophilized polyalkylene oxide-asparaginase composition can also include producing an aqueous concentrated composition that is subsequently lyophilized. A process flow diagram for producing an aqueous concentrated composition is shown in Figure 1. As shown in Figure 1, the method for producing the aqueous concentrated composition can include one or more of the following steps: preparing a solution of asparaginase (e.g., L-asparaginase) (10); conjugating an oxide (e.g., polyethylene glycol) to asparaginase (20); purifying the polyalkylene oxide-asparaginase (30); filtering and concentrating the polyalkylene oxide-asparaginase solution (40); diluting the polyalkylene oxide-asparaginase solution (50); filtering the polyalkylene oxide-asparaginase solution and filling the polyalkylene oxide-asparaginase solution into sterile containers (60); and storing the polyalkylene oxide-asparaginase solution (70).

[0143] In the method for producing an aqueous concentrate composition, asparaginase (e.g., L-asparaginase) A solution of asparaginase (asparaginase) can be prepared. The asparaginase can be mixed with a solution, such as an aqueous solution (e.g., a buffered aqueous solution). Examples of suitable buffers include, but are not limited to, phosphate buffer, phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), Tris buffer, Ringer's lactate buffer, borate buffer, and combinations thereof. In some cases, the asparaginase is mixed with a phosphate buffer.

[0144] In some embodiments, the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate. In some cases, the amount of dibasic sodium phosphate in the aqueous concentrate composition ranges from 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.1 to 0.4 wt.%, or 0.2 to 0.4 wt.%, or 0.2 to 0.3 wt.%, or 0.25 to 0.3 wt.%. For example, dibasic sodium phosphate can be present in the aqueous concentrate composition in an amount ranging from 0.1 to 0.5 wt.%. In certain embodiments, the amount of monobasic sodium phosphate in the aqueous concentrate composition is 0.01-0.9 wt.%, or 0.01-0.8 wt.%, or 0.01-0.7 wt.%, or 0.01-0.6 wt.%, or 0.01-0.5 wt.%, or 0.01-0.4 wt.%, or 0.01-0.3 wt.%, or 0.01 % or 0.02-0.09 wt.%, or 0.03-0.08 wt.%, or 0.04-0.08 wt.%, or 0.045-0.075 wt.%, or 0.04-0.07 wt.%, or 0.05-0.07 wt.%. For example, the monobasic sodium phosphate can be present in the aqueous concentrate composition in an amount ranging from 0.01 to 0.1 wt.

[0145] Additional components that may be included in the aqueous concentrate composition include salts. Examples of suitable salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and the like, and combinations thereof. In a particular example, the salt is sodium chloride.

[0146] In some cases, the amount of salt (e.g., sodium chloride) in the aqueous concentrate composition is in the range of 0.05 to 1 wt.%, such as 0.1 to 0.9 wt.%, or 0.1 to 0.8 wt.%, or 0.1 to 0.7 wt.%, or 0.1 to 0.6 wt.%, or 0.1 to 0.5 wt.%, or 0.2 to 0.5 wt.%, or 0.3 to 0.5 wt.%, or 0.4 to 0.5 wt.%, or 0.4 to 0.45 wt.%. For example, the salt (e.g., sodium chloride) can be present in the aqueous concentrate composition in an amount in the range of 0.1 to 1 wt.%.

[0147] Another component that may be included in the aqueous concentrate composition is a sugar. Examples of suitable sugars include, but are not limited to, sucrose, mannitol, maltose, trehalose, 2-hydroxypropyl-β-cyclodextrin (HPCD), lactose, glucose, fructose, galactose, and the like. Examples of sugars include sucrose, glucosamine, and the like, and combinations thereof. In particular examples, the sugar is a disaccharide. For example, the disaccharide may be sucrose.

[0148] In some cases, the amount of sugar (e.g., sucrose) in the aqueous concentrate composition is 0.5 to 20 wt.%, or 1 to 15 wt.%, or 1 to 10 wt.%, or 1 to 9 wt.%, or 1 to 8 wt.%, or 2 to 7 wt.%, or 2 to 6 wt.%, or 3 to 5 wt. % or 4-5 wt.%. For example, sugar (e.g., sucrose) can be present in the aqueous concentrate composition in an amount ranging from 1 to 10 wt.%.

[0149] After preparation of the asparaginase solution, the asparaginase can be conjugated to a polyalkylene oxide (e.g., polyethylene glycol) so that the polyalkylene oxide is covalently bound to the asparaginase to form a polyalkylene oxide-asparaginase conjugate. After preparation of the polyalkylene oxide-asparaginase, the solution can be subjected to purification. In some cases, purification involves passing the solution through a filter to remove certain substances from the solution. The filtration step can produce a substantially purified polyalkylene oxide-asparaginase.

[0150] In some examples, the filtered polyalkylene oxide-asparaginase solution is then subjected to diafiltration and concentration steps. The polyalkylene oxide-asparaginase solution can be diafiltered using an ultrafiltration membrane, and the resulting polyalkylene oxide-asparaginase concentrate can be obtained. The concentrate from the diafiltration step can then be diluted so that the solution contains the desired concentration of polyalkylene oxide-asparaginase. Suitable buffers useful for the dilution step include those mentioned above. In certain cases, a phosphate buffer is used to dilute the polyalkylene oxide-asparaginase solution, thus producing the desired aqueous concentrated composition. For example, the concentrate from the diafiltration step can be diluted to contain an amount of polyalkylene oxide-asparaginase such that the resulting aqueous concentrated composition has a potency (activity) in the range of 100-5,000 IU / mL, such as 500-4,500 IU / mL, or 500-4,000 IU / mL, or 500-3,500 IU / mL, or 500-3,000 IU / mL, or 1,000-3,000 IU / mL, or 1,500-3,000 IU / mL. In certain examples, the aqueous concentrated composition contains an amount of polyalkylene oxide-asparaginase in the range of 1,500-3,000 IU / mL. In some cases, the amount of polyalkylene oxide-asparaginase in the aqueous concentrated composition is greater than the amount of polyalkylene oxide-asparaginase in the reconstituted lyophilized compositions described herein. In some cases, the diafiltration produces a substantially purified polyalkylene oxide-asparaginase.

[0151] This aqueous concentrate composition can then be filtered and filled into a sterile container. Examples of suitable container materials include, but are not limited to, polymers such as polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyether ether ketone (PEEK), and polystyrene. For example, this can be a sterile polymer bag. This aqueous concentrate composition can be stored in the container for a certain period of time and processed into a freeze-dried, shelf-stable composition of the present disclosure.

[0152] An embodiment of the method may further include shipping the aqueous concentrate composition to a remote location. A "remote location" is a location different from the location where the aqueous concentrate composition is manufactured. For example, a remote location can be another location within the same city (e.g., an office laboratory, etc.), another location in a different city, another location in a different state, another location in a different country, etc. When one item is described as being "remote" from another, what is meant is that the two items are in the same room but separated, or at least in different rooms, or in different buildings, and can be at least 1 mile, 10 miles, or 100 miles or more apart.

[0153] In certain embodiments, as described above, the method includes lyophilizing the aqueous concentrated composition in a manner sufficient to produce a lyophilized storage-stable polyalkylene oxide-asparaginase composition. In some instances, this lyophilization can occur in a unit-dose container. Lyophilizing the aqueous concentrated composition to produce a lyophilized storage-stable polyalkylene oxide-asparaginase composition in the unit-dose container facilitates production of the lyophilized composition in the unit-dose container, for example, by eliminating the need to lyophilize the aqueous concentrated composition in a separate container and then transfer the lyophilized composition from the separate container to the unit-dose container. As such, in some embodiments, the method includes introducing the aqueous concentrated composition into the unit-dose container and lyophilizing the aqueous concentrated composition in the unit-dose container. As described above, the unit-dose container may be a vial, such as a glass vial.

[0154] After lyophilization, the method can further include sealing the lyophilized composition in a unit dose container. For example, a seal or lid is attached to the opening of the unit dose container, thus enclosing the unit dose container. The sealed container can be stored for an extended period of time, such as 1 week or more, or 2 weeks or more, or 3 weeks or more, or 1 month or more, or 2 months or more, or 3 months or more, or 4 months or more, or 6 months or more, or 9 months or more, or 1 year or more, or 1.5 years (e.g., 18 months) or more, or 2 years or more, or 2.5 years (e.g., 30 months) or more, or 3 years or more, or 3.5 years (e.g., 42 months) or more, or 4 years or more, or 4.5 years (e.g., 54 months) or more, or 5 years or more. For example, a long period of time can be 6 months or more. In some cases, the sealed container can be stored for 9 months or more. In some cases, the sealed container can be stored for 1 year (e.g., 12 months) or more. In some cases, the sealed container can be stored for 1.5 years (e.g., 18 months) or more. In some cases, the sealed container can be stored for 2 years (eg, 24 months) or more.

[0155] [kit] Also provided are kits containing one or more of the above liquid and / or lyophilized compositions for use in practicing the subject methods. For example, the kits include a unit-dose container containing a liquid composition described herein. Or, for example, the kits include a unit-dose container containing a lyophilized composition described herein. In some examples, the kits include two or more unit-dose containers, each containing a liquid composition described herein. In some examples, the kits include two or more unit-dose containers, each containing a lyophilized composition described herein. In some examples, the kits include two or more unit-dose containers, one or more of the unit-dose containers containing a liquid composition described herein and one or more of the unit-dose containers containing a lyophilized composition described herein. In certain embodiments, the kits include packaging configured to contain the unit-dose containers. The packaging can be a sealed packaging, such as a sterile, sealed packaging. Sterile packaging can be configured to be sealed from the outside environment so that the packaging is substantially free of pathogens (fungi, bacteria, viruses, spores, etc.) within the packaging. In some examples, the packaging is optionally sealed under an airtight and / or vacuum seal with water vapor resistant packaging, etc.

[0156] In certain embodiments, the kit includes a buffer. For example, the kit can include a diluent liquid, e.g., a diluent buffer, suitable for administration to a subject, etc. The kit can further include other components, e.g., administration devices, fluid sources, etc., that find use in practicing the subject methods. The various components in the kit can be packaged as desired, e.g., together or separately. Components of the subject kits can be in separate containers, or multiple components can be in a single container, and the container and / or packaging materials (or portions thereof) of the kit can be sterile, if desired.

[0157] In addition to the above-mentioned components, the subject kits can further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are typically recorded on a suitable recording medium. For example, the instructions can be printed on paper, plastic, or the like. As such, the instructions can be present in the kit as a package insert, on a label on the kit container, or on a component thereof (i.e., associated with the packaging or part of the packaging). In other embodiments, the instructions are stored electronically as a data file, e.g., on a portable flash drive, CD-ROM, On a suitable computer-readable storage medium such as DVD-ROM, Blu-ray, etc. In some embodiments, the actual instructions are not present in the kit, but rather directions for obtaining the instructions are provided from a remote source, e.g., via the internet. An example of this embodiment is a kit that includes a URL where the instructions can be viewed and / or downloaded. The instructions can be downloaded. Along with the instructions, an embodiment for obtaining the instructions is also recorded on a suitable medium.

[0158] [utility] The subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods find use where there is a desire to treat a potentially treatable disease or condition in a subject by administration of polyalkylene oxide-asparaginase. For example, the subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods find use in treating a neoplastic condition in a subject. In some cases, the subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods find use in treating cancer in a subject. Examples of types of cancer that may be treatable using the subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods include, but are not limited to, leukemias such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Thus, the subject lyophilized storage-stable compositions and methods find use in providing therapeutically effective treatments for neoplastic conditions such as cancer, including, but not limited to, leukemias such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

[0159] The lyophilized, storage-stable compositions and methods of the present disclosure find application in treating subjects of any age. In some cases, the subject compositions (e.g., liquid or lyophilized, storage-stable compositions) and methods find application in treating adults. For example, adult human subjects can be 18 years of age or older. In other cases, the compositions (e.g., liquid or lyophilized, storage-stable compositions) and methods find application in treating juveniles. For example, juvenile human subjects can be under 18 years of age.

[0160] The compositions and methods of the present disclosure find use in applications where a shelf-stable composition is desired. For example, the compositions and methods of the present disclosure find use in providing shelf-stable compositions that are stable for extended periods of time (e.g., do not substantially decompose and / or retain substantially all of their activity). For example, the compositions and methods of the present disclosure find use in providing shelf-stable compositions that are stable for extended periods of time, such as 1 week or more, or 2 weeks or more, or 3 weeks or more, or 1 month or more, or 2 months or more, or 3 months or more, or 4 months or more, or 6 months or more, or 9 months or more, or 1 year or more, or 1.5 years (e.g., 18 months) or more, or 2 years or more, or 2.5 years (e.g., 30 months) or more, or 3 years or more, or 3.5 years (e.g., 42 months) or more, or 4 years or more, or 4.5 years (e.g., 54 months) or more, or 5 years or more. In some cases, the compositions and methods of the present disclosure find use in providing shelf-stable compositions that are stable for 9 months or more. In some cases, the compositions and methods of the disclosure find use in providing shelf-stable compositions that are stable for 1 year (e.g., 12 months) or more. In some cases, the compositions and methods of the disclosure find use in providing shelf-stable compositions that are stable for 1.5 years (e.g., 18 months) or more. In certain embodiments, the compositions and methods of the disclosure find use in providing shelf-stable compositions that are stable for 2 years (e.g., 24 months) or more. In certain embodiments, the compositions and methods of the disclosure find use in providing shelf-stable compositions that increase the shelf life of the composition by up to 1 week, or 2 weeks, or 3 weeks, or 1 month, or 2 months, or 3 months, or 4 months, or 6 months, or 9 months, or 1 year, or 1.5 years (e.g., 18 months), or 2 years, or 2.5 years (e.g., 30 months), or 3 years, or 3.5 years (e.g., 42 months), or 4 years or more, or 4.5 years (e.g., 54 months), or 5 years. In certain embodiments, the compositions and methods of the disclosure find use in providing shelf-stable compositions that increase the shelf life of the composition by 1 month to 5 years, or 6 months to 4 years, or 9 months to 3 years, or 1 year to 2 years.

[0161] In certain embodiments, the dose of the present disclosure can be administered prior to, simultaneously with, or subsequent to one or more other oncologic treatments for the treatment of related or unrelated conditions. When administered simultaneously with the other oncologic treatments, this is achieved by administration of a pharmaceutical composition with at least one other active agent, such as a chemotherapeutic agent, that provides a therapeutically effective dose in combination according to a specific therapeutic regimen. The administration of separate pharmaceutical compositions or treatments can occur simultaneously or at different times (e.g., sequentially, in either order, on the same day, or on different days), so long as the combination of these agents produces a therapeutically effective effect in the subject being treated. Thus, the methods and compositions of the present disclosure find use in treating subjects using combination therapies that include administration of a polyalkylene oxide-asparaginase of the present disclosure in combination with one or more additional active agents and / or treatments (e.g., radiation, chemotherapy, immunotherapy, etc.).

[0162] As can be seen from the disclosure provided above, embodiments of the present disclosure have a wide variety of applications. Accordingly, the examples provided herein are provided for illustrative purposes and are not intended to be construed as limiting in any way to embodiments of the present disclosure. Those skilled in the art will recognize that various non-essential parameters can be changed or modified to produce essentially the same results. Therefore, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use embodiments of the present disclosure, and are not intended to limit the scope of what the inventors contemplate their invention, or to represent that all, or only, of the experiments below were performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be expected. Unless otherwise indicated, parts are by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0163] The following examples are provided for illustrative purposes only and are not intended to limit the embodiments of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. [Example]

[0164] Example 1 Concentrated polyethylene glycol-asparaginase with SS-PEG linker A concentrated bulk composition was prepared according to the following procedure: Following preparation of this concentrated bulk composition, a lyophilized composition was prepared from the concentrated bulk composition according to the procedure described below: Figure 1 shows a process flow diagram for a method of making a lyophilized storage-stable composition according to an embodiment of the present disclosure.

[0165] Preparation of L-asparaginase solution The amount of L-asparaginase required for the treatment is calculated and placed in a 6 L stainless steel beaker. The L-asparaginase was weighed into the flask and mixed for 5-10 minutes. The amount of phosphate buffer needed to dilute to 1 mL was calculated and weighed. Paraginase was then added to the phosphate buffer and mixed for 10-15 minutes in a 7-gallon stainless steel pressure vessel. Samples were removed for protein and specific activity testing.

[0166] PEGylation The amount of SS-PEG needed for this step was calculated and weighed. The 7-gallon stainless steel pressure vessel containing the diluted L-asparaginase solution was heated to 29-31°C under gentle agitation. Once the L-asparaginase solution reached the appropriate temperature range, the mixer speed was increased. The drop pump was started and the pH was adjusted to 7.7-7.9 with 0.5 N NaOH. The SS-PEG was added to a 7-gallon stainless steel pressure vessel. After 30 minutes, The drip pump was stopped and the temperature jacket was removed.

[0167] purification The process material was then filtered using a diafiltration peristaltic pump through a 0.45 μm filter (Millipore, Billerica, Mass.) into another 7-gallon stainless steel pressure vessel. After clarification, phosphate buffer was added to the original 7-gallon stainless steel pressure vessel and pumped into the 7-gallon stainless steel pressure vessel through the 0.45 μm filter as a rinse.

[0168] Diafiltration / Concentration The amount of PBS required for 15X diafiltration was calculated, and the diafiltration system was activated. The 7-gallon stainless steel pressure vessel was placed on a balance. A Millipore Pellicon®-2 diafiltration system was set up with a diafiltration peristaltic pump and a 100,000 Da nominal molecular weight limit membrane pretreated with 5 L of PBS. The level control system, including the buffer peristaltic pump, was then activated, and the 7-gallon stainless steel pressure vessel was placed on the balance. For conditioning, the Pellicon®-2 system was used. The mixture was filled with polyethylene glycol-asparaginase and recirculated for 5 minutes. Following diafiltration, the permeate waste line was opened and diafiltration was initiated. After 15 and 30 minutes, samples were obtained from the permeate waste line into vials and submitted for activity and protein testing. The material intended for lyophilization was diafiltered and then concentrated to ≥18.0 mg / mL and ≥1,850 IU / mL. Upon completion of diafiltration, the volume of material in process in the 7-gallon stainless steel pressure vessel was adjusted by removing the appropriate amount of permeate to reach the desired concentration (≥18.0 mg / mL for processing on lyophilized formulations). PBS was then pumped through the system into the 7-gallon stainless steel pressure vessel as a rinse.

[0169] Quality control assays of the diafiltrated product were performed, including determination of the impurity profile of the product. To confirm product retention by the diafiltration membrane, permease enzyme activity (EEA) was measured after 15 and 30 minutes. Free PEG and N-hydroxysuccinimide (NHS) were measured in the final product. and were components of the process-related impurity profile. The official in-process controls for the diafiltration unit operation are shown in Table 1 below. From three drug substance compositions (e.g., concentrated bulk drug substance compositions) for lyophilization The NHS and free PEG data generated is shown below in Table 2. The data generated demonstrates that small changes in the diafiltration / concentration process do not affect the quality of the product.

[0170] [Table 1]

[0171] [Table 2]

[0172] Dilution The in-process material was mixed in a 7-gallon stainless steel pressure vessel before samples were removed for activity and protein testing. The in-process material volume was then diluted with PBS to a target protein concentration of 2:18.0 mg / mL (target 20.0 mg / mL) for drug substance intended for lyophilized compositions and 2:1850 IU / mL activity for drug substance. This diluted in-process material was mixed before samples were removed for quality assurance testing (see Table 1).

[0173] Sterile filtration The concentrated solution for lyophilization was filtered through a 0.2 μm filter into a disposable 20 L sterile bag for storage until lyophilization. Samples were collected and subjected to sterility testing. Bulk drug substance compositions containing ligninase can be stored in 20 L bags at 2-8°C for up to 2 months before lyophilization.

[0174] Container closure For this lyophilization, the bulk drug substance containing polyethylene glycol-asparaginase The drug substance composition was processed into a lyophilized composition following a 0.2 μm filtration step and delivered into a pre-sterilized, disposable 20 L bioprocess bag. The bag's construction materials included a layer of low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), nylon, and ethylene vinyl alcohol (EVOH). The surface in direct contact with the product (inner layer) was LDPE. Each bag had two openings with tubing secured to the bag, which received the bulk of the polyethylene glycol-asparaginase-containing drug substance composition for lyophilization. The bags were irradiated and opened using a 25-40 kGy exposure and subjected to Limulus Amebocyte Lysate (LAL) endotoxin testing, 100% visual seal and air leak testing, as well as 100% visual inspection after assembly. Qualification testing showed that the inner layer of the bags met USP <88> Class IV Plastics Test, USP <87> Cytotoxicity Testing, and USP <661> The bags have passed biological reactivity tests, including tests for the physicochemical properties of the bags. <788> The instructions for particular matter in injections were followed and all extractables testing complied with manufacturer requirements.

[0175] Drug substance stability waiting time The concentrated bulk drug substance was monitored at 2-8°C for 0, 2, 4, 6, 8, and 12 weeks. Stability samples were held in 250 mL polyethylene sample bags with product-contact surfaces. Data from the study design are presented in Table 3 for Lot 1, Table 4 for Lot 2, and Table 5 for Lot 3. Stability data for the concentrated bulk drug substance for the lyophilized composition met acceptance criteria throughout the 12-week testing plan and demonstrated a 2-month waiting period from concentrated bulk drug substance manufacturing and manufacturing of the lyophilized composition was acceptable.

[0176] [Table 3]

[0177] [Table 4]

[0178] [Table 5]

[0179] Lyophilized Composition The lyophilized powder composition for injection contains 3,750 IU of active PEGylated L-asparaginase. The lyophilized composition was manufactured in single-use vials containing 4.5% sucrose, dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride after reconstitution with 5.2 mL of WFI. The components of the lyophilized composition after reconstitution were provided in Table 6. In addition, the lyophilized composition had a 20 mm aluminum flip-off seal. It was provided in a treated glass vial container (Nipro Type 1).

[0180] [Table 6]

[0181] Formulation development of lyophilized polyethylene glycol-asparaginase The objectives of developing a lyophilized polyethylene glycol-asparaginase formulation were to: The objective was to achieve a stable lyophilized composition suitable for storage at 2-8°C or 25°C for 18 months.

[0182] To evaluate the feasibility of a polyethylene glycol-asparaginase lyophilized composition Initial experiments were conducted to evaluate the effectiveness of the freeze-dried asparaginase. Five variations of the freeze-dried composition were investigated. Each formulation contained 5 mg / mL polyethylene glycol-asparaginase and 5% w / v of one of five different cryoprotectants (mannitol, Maltose, sucrose, trehalose, and / or -HPCD). In addition, polyethylene glycol-asparaginase without cryoprotectant was also subjected to lyophilization. These six lyophilized compositions were compared with the polyethylene glycol-asparaginase (Oncaspar) in liquid formulation used to prepare these different formulations. The results of the study are shown in Table 1. As can be seen from Figure 7, sucrose was converted to polyethylene glycol-asparagine during freeze-drying. The purity of the lyophilized composition containing 5% sucrose (83.0%) was found to be the most effective in preserving the purity of the enzyme. The purity of the liquid polyethylene glycol-asparaginase formulation (83.8%) was comparable to that of the liquid polyethylene glycol-asparaginase formulation used to prepare six formulations for subsequent lyophilization. It was comparable.

[0183] [Table 7]

[0184] Several different additives (e.g., sucrose, trehalose, mannitol, and polysorbate 80) were evaluated as potential stabilizing agents for inclusion in the lyophilized compositions. Four experiments were conducted, each containing four different compositions. Small-scale pilot-scale batches of lyophilized polyethylene glycol-asparaginase were prepared. were prepared with different excipients and the stability was evaluated. A list of the formulations tested is shown in Table 8.

[0185] [Table 8]

[0186] All lots listed in Table 8 were evaluated in stability studies at 5°C, 25°C and 40°C and passed a panel of tests (activity, specific activity, protein, pH, purity (GF-HPLC) ), aggregates (GF-HPLC), and microparticles), which The key quality attributes of lenglycol-asparaginase at release and stability were evaluated. Based on the stability data collected from 16 different formulations listed in Table 8, sucrose was identified as a suitable cryoprotectant (eg, stabilizer). Various sucrose concentrations were tested to evaluate the appropriate concentration for a more robust (i.e., less impactful to the lyophilization procedure) and more stable product. Additional pilot-scale batches containing different concentrations of sucrose were prepared, as summarized in Table 9. The sucrose and PEG-asparaginase concentrations shown in Table 9 were determined based on the concentration of the concentrated bulk product. The amounts shown indicate the amounts present in the agent raw material. During the lyophilization process, the vials are filled to 2.5 mL before the start of lyophilization. These lyophilized vials are reconstituted with 5.0 mL, resulting in a final sucrose and PEG-asparaginase content that is approximately half of that shown in the table. This study demonstrated that increasing the sugar content allows for higher freeze-drying temperatures, which puts less stress on the freeze-dried product and also reduces the overall freeze-drying cycle time, and results in a drier (more stable) freeze-dried product.

[0187] [Table 9]

[0188] The five lots listed in Table 9 were also evaluated in stability studies at 5°C, 25°C, and 40°C. Activity, specific activity, protein, pH, purity (GF-HPLC), aggregation Quality attributes such as total mass (GF-HPLC), and particulates were evaluated during this stability study. Purity and potency results from accelerated (25°C) and stressed (40°C) stability studies for these lots are shown in Figures 2-5. These stability data indicated that the formulation containing 10% sucrose (5% sucrose after reconstitution of the final product with 5 mL / vial of WFI) provided the product with the highest stability in terms of purity and potency. Other quality attributes were less affected by the different formulations and were again stable in the lots with 10% sucrose. Figure 2 shows the purity and potency of the lyophilized PEG-asparaginase compositions of lots SA, SB, SC, SD, SE, and IA at 40°C. Figure 2 shows a graph of potency (%) versus time (weeks) for lyophilized PEG-asparaginase compositions of lots SC, SD, SE, and IA at 40°C. Figure 4 shows a graph of purity (%) versus time (weeks) at 25°C for lyophilized PEG-asparaginase compositions of lots SA, SB, SC, SD, SE, and IA. Figure 5 shows the results of freeze-dried PEG-Asp of lots SC, SD, SE, and IA. 1 shows a graph of potency (IU / mL) versus time (weeks) at 25°C of paraginase compositions.

[0189] Freeze drying A buffer solution containing water for injection (WFI), dibasic sodium phosphate, monobasic sodium phosphate, sodium chloride, and sucrose was prepared and used for dilution of the concentrated bulk drug substance.

[0190] 90% of the desired weight of WFI needed to create the buffer solution was added to a beaker. Monobasic sodium phosphate, dibasic sodium phosphate, and sodium chloride were then weighed individually, added to WFI, and mixed until dissolved. The required amount of sucrose was weighed and added to the combined buffer solution and mixed until dissolved. The solution's pH was then measured and adjusted to 7.3 ± 0.1 by slow addition of NaOH. WFI was added to the buffer solution as needed, and the density of the concentrated bulk solution was used to weigh the required volume of concentrated bulk solution needed for that batch size. A sample of the buffer was collected for sucrose assay. This concentrated bulk solution was added to the buffer solution, and the final solution was mixed for at least (not less than) 10 minutes. Once mixing was complete, confirmatory pH measurements (7.3 ± 0.1) were taken from the top, middle, and bottom of the container, and samples were collected for in-process testing of protein, density, sucrose, and bioburden before filtration.

[0191] Following final formulation, the solution underwent sterile filtration. The filtration tubing assembly was placed into a formulated bulk container, and the bulk was then filtered through two 0.22 μm filters placed on the filtration tubing assembly into a pre-sterilized, ready-to-fill 10 L bioprocess bag. Once all product had been transferred to the 10 L bioprocess bag, the filtration tubing assembly was removed from the bioprocess bag, and the filter was tested to ensure integrity. One 20 mL pre-lyophilization sample was collected from upstream (unfiltered) of the filter and tested according to the final drug product specifications.

[0192] A flow diagram illustrating the final formulation and sterile filtration processing steps is shown in the figure.

[0193] Aseptic filling and freeze drying Following completion of sterile filtration, the 10 L biobag containing the bulk drug substance solution was attached to a fill tubing assembly and product vials were then filled to a target fill weight of 2.5 g / vial and partially stoppered using a Flexicon FMB210 filler. Fill weights were monitored during the run by performing a minimum of one weight check (one vial) per filled tray (processing limit: 2.43-2.57 g, warning limit: 2.38-2.62 g). When the filling run was completed, 20 pre-lyophilized vials were tested and all remaining filled vials were transferred to stainless steel lyophilization trays and subsequently filled into a pre-chilled (5°C) 270 ft² Hull freeze-drying system for lyophilization. The phases that can be detected are shown in Table 10.

[0194] [Table 10]

[0195] After the lyophilization cycle is complete, the vent chamber is backfilled with nitrogen gas and the vials are fully stoppered. A flow diagram showing the aseptic filling and lyophilization process steps is shown in Figure 7. The fully stoppered vials are packaged in cartons (90 vials / carton) for subsequent storage and / or shipping.

[0196] Specifications of the lyophilized composition Product specifications for the lyophilized compositions are shown in Table 11.

[0197] [Table 11]

[0198] Lot Analysis Lot analyses of the lyophilized composition product lots are provided below in Table 12. All three The results of this lot were within the release specifications.

[0199] [Table 12]

[0200] stability studies Lyophilized drug product lots 1, 2, and 3 were placed into long-term (2-8°C) and accelerated (25±3°C; 60%±5%RH) stability studies. These lots were also placed into heat stress stability studies (40±2°C; 75%±5%RH) for evaluation of the product's heat-induced degradation profile.

[0201] Long term stability (2~8°C) Stability data generated for lyophilized drug product lots stored under long-term (2-8°C) conditions are provided in Tables 13-16. Long-term stability data demonstrated that lyophilized drug product stored at 5±3°C remained well within the acceptance criteria for stability at all time points. Moisture content (KF) data was measured over 12 weeks at 2-8°C storage conditions, ranging from 0.96% to 1.35% (specification = NMT 3.0%). Unlike commercially available liquid drug products, which have demonstrated increased activity and decreased purity over time, this trend was not observed with the lyophilized compositions. Stability graphs for purity (Figure 8) and potency (Figure 9) as well as aggregates (Figure 10) at 2-8°C are shown in the accompanying figures.

[0202] [Table 13]

[0203] [Table 14]

[0204] [Table 15]

[0205] [Table 16]

[0206] Accelerated stability (25±3°C; 60%±5%RH) Stability data for lyophilized drug product lots stored under accelerated (25±3°C; 60%±5% RH) conditions are provided in Tables 17-20. The stability data indicated that the lyophilized drug product stored under accelerated conditions remained well within the acceptance criteria at all stability time points. Moisture content (KF) data was measured at 25°C storage conditions and ranged from 1.12% to 1.23% (specification = NMT 3.0%) over 4 weeks. Stability charts at 25±3°C are shown in the accompanying figures for the quality attributes purity (Figure 11), potency (Figure 12), as well as aggregates (Figure 13).

[0207] [Table 17]

[0208] [Table 18]

[0209] [Table 19]

[0210] [Table 20]

[0211] Heat stress stability (40±2°C; 75%±5%RH) Stability data for lyophilized drug product lots stored under stressed (40±2°C; 75%±5% RH) conditions are provided in Tables 21-24. The stability data indicated that the lyophilized drug product stored under stressed conditions remained well within the acceptance criteria for the duration of the study. Moisture content (KF) data was measured over 4 weeks at 40°C storage conditions, ranging from 1.16% to 1.45% (specification = NMT 3.0%). Stability charts at 40±2°C for potency (Figure 14), purity (Figure 15), as well as aggregates (Figure 16) are shown in the accompanying figures.

[0212] [Table 21]

[0213] [Table 22]

[0214] [Table 23]

[0215] [Table 24]

[0216] Example 2 Polysaccharides with SC-PEG linkers (i.e., succinimidyl carbonate linkers) Compositions containing triethylene glycol-asparaginase, pegaspargase pegol ( Succinimidyl carbonate-polyethylene glycol [SC-PEG] (E. coli L-asparaginase) was prepared. The components of this composition are provided in Table 25.

[0217] [Table 25]

[0218] Example 3 Following preparation of the concentrated bulk composition, a lyophilized composition of pegaspargase pegol can be prepared from the concentrated bulk composition. Figure 1 shows an example of a process flow diagram that can be used to prepare a lyophilized, shelf-stable composition according to an embodiment of the present disclosure. This lyophilized composition powder for injection can be produced in single-use vials containing 3,750 IU of activity of pegaspargase pegol (750 IU / mL after reconstitution with 5.2 mL of WFI). The components of this lyophilized composition after reconstitution include 4.5% sucrose, dibasic sodium phosphate, monobasic sodium phosphate, and sodium chloride. The components of the lyophilized composition are provided in Table 26.

[0219] [Table 26]

[0220] Example 4 The purpose of this study was to provide comparative information on the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of liquid pegaspargase (PEG-L-asparaginase; Oncaspar®) and lyophilized pegaspargase when administered intravenously by slow bolus infusion to Beagle dogs once (day 1) or once weekly for 4 weeks (days 1, 15, 22, 29, and 36). Because reconstituted lyophilized pegaspargase is administered intravenously to humans, the same route of administration was used in this study. Single-dose and repeat-dose PK / PD studies were required to determine and compare the pharmacokinetics and pharmacodynamics of the liquid and reconstituted lyophilized versions at equivalent doses.

[0221] Beagle dogs (nominally 5 / sex / group) were administered 500 IU / kg liquid pegaspargase or reconstituted lyophilized pegaspargase via intravenous injection at a dosage of 0.667 mL / kg (see Table 27). The beagles were approximately 6 months of age, males weighing 7.2-10.7 kg, and females weighing 5.6-8.2 kg.

[0222] Liquid pegaspargase (pH 7.2-7.45 mL of PBS buffer containing 50 mM phosphate and 0.85% saline) was used as supplied, with no preparation required. Lyophilized pegaspargase (see Example 1) was prepared for administration (using a 21-gauge syringe) by reconstituting the vial contents with 5.2 mL of water for injection (WFI) using aseptic technique to achieve a concentration of 750 IU / mL. Prior to administration, the vial contents were gently swirled until thoroughly mixed. The mixture was visually inspected for particulate issues, such as cloudiness or discoloration. A fresh formulation was prepared each day of dose administration, kept at room temperature, and used within 2 hours of preparation.

[0223] [Table 27]

[0224] Animals in groups 1 and 3 received a single dose of liquid pegaspargase or lyophilized pegaspargase, respectively, on day 1. Animals in groups 2 and 4 received repeated doses of liquid pegaspargase or lyophilized pegaspargase, respectively. A slow bolus (over approximately 2 minutes) intravenous injection was administered within 2 hours of test article preparation. An indwelling catheter (non-butterfly catheter) was used, and the catheter cap was then cleaned of any residual dose by flushing with saline. A straight needle was inserted into the catheter cap to ensure needle placement remained consistent for the 2-minute duration.

[0225] Blood samples were obtained from all animals on days 1 and 36 for pharmacokinetic and pharmacodynamic analyses. Approximately 1.0 mL of whole blood was obtained at each time point. Animals were not anesthetized or fasted prior to blood collection. Blood was collected into tubes containing sodium heparin anticoagulant and placed upright in ice water. Blood samples were centrifuged for 5 minutes (approximately 3000 rpm, approximately 4°C) to obtain plasma within 5 minutes of the start of blood sample collection.

[0226] PD: One 125 μL aliquot of plasma was pipetted into a cryotube prefilled with 125 μL of SeraPrep for asparagine determination. The tube was inverted for 3 h to mix with the SeraPrep and immediately flash-frozen in liquid nitrogen or methanol / dry ice within 15 min of collection. All SeraPrep-containing aliquots were analyzed for asparagine by high-performance liquid chromatography (HPLC) with mass spectrometric detection (LC-MS / MS). Analyzed for paragine determination.

[0227] PK: The remainder of the plasma was divided into two cryotubes for asparaginase activity determination and flash-frozen within 30 minutes of blood sample collection. All aliquots not containing SeraPrep were analyzed for asparaginase activity by a colorimetric mixed enzyme reaction.

[0228] result Analysis performed on the lyophilized pegaspargase during the treatment period determined that the appropriate concentration (for batch analysis; expected protein concentration of 6.6 mg / mL and activity of 741 IU / mL) of the dosage formulation was administered (Table 28).

[0229] [Table 28]

[0230] Pooled group mean plasma concentrations of asparaginase on Day 1 (Cmax) and pooled group mean area under the plasma asparaginase concentration-time curve (AUC) on Day 1 estimated up to 552 hours post-dose. 0.552 ), as well as their values ​​following repeat dosing on day 36, are summarized for each group (males and females combined) in Table 29.

[0231] [Table 29]

[0232] The study was designed as a parallel group design, and the data were statistically analyzed using analysis of variance techniques. Cmax and AUC from both days 0.552 Data were analyzed by an ANOVA model with formulation, time, sex and their interactions as factors. These two pegaspargase formulations had similar Cmax and AUC 0.552 The geometric mean ratios were analyzed in terms of the mean mean and the corresponding two-sided 90% confidence intervals are summarized in Table 30.

[0233] [Table 30]

[0234] For Cmax, there was evidence of bioequivalence because the confidence interval (0.867-0.970) was included in the rejection region. AUC 0.552 For the confidence interval (0.841 There was evidence of bioequivalence because the critical region included the mean (~0.996).

[0235] The mean maximum plasma concentration (Cmax) of pegaspargase and the pooled mean area under the plasma asparaginase concentration-time curve (AUC) estimated up to 552 hours after dosing on Day 1 were 0.552 ), and their values ​​following repeat dosing on day 36 are summarized in Table 31 below for each group (by sex) with standard deviations in parentheses.

[0236] [Table 31]

[0237] The mean maximum plasma concentration (Cmax) of asparaginase and the area under the plasma asparaginase concentration-time curve (AUC) for the lyophilized formulations were 0.552 ) relationships and dose levels are expressed as a ratio compared to the liquid formulation and are presented in Table 32.

[0238] [Table 32]

[0239] Cmax and AUC of systemic exposure to asparaginase in dogs 0.552 The AUC values ​​were similar for the reconstituted lyophilized pegaspargase compared to the liquid formulation on Day 1 and subsequent repeat dosing on Day 36. The AUC values ​​were similar for the two formulations. 0.552 There was no evidence that the Cmax values ​​were significantly different, but there was some evidence that the Cmax values ​​differed between the different formulations, with the Cmax being slightly lower (8%) when administered as the lyophilized product than when administered as a liquid formulation.

[0240] Cmax and AUC of systemic exposure to asparaginase in female dogs 0.552 The values ​​of are generally similar to those of exposure in males, and Cmax or AUC 0.552 There was no evidence of statistically significant sex-related differences in systemic exposure.

[0241] Other parameters assessed during the study were: viability, clinical observations, body weight, food consumption, respiratory rate, body temperature, hematology, coagulation and blood chemistry; for these, no adverse effects related to the test article were observed. Cmax values ​​and range (AUC ) of systemic exposure to asparaginase in dogs after repeated intravenous administration (day 36) 0.552) was higher than the value after a single dose (day 1), and these differences This was statistically significant (p<0.001). AUC 0.552 The mean cumulative ratio calculated based on the values ​​(note that different animals provided data on each day) was greater than indicated that asparaginase accumulation occurred after repeated intravenous administration of liquid pegaspargase.

[0242] Overall, these two pegaspargase formulations exhibited similar Cmax and AUC values, as the corresponding two-sided 90% confidence intervals for the geometric mean ratios fell entirely within the conventional margins of bioequivalence, ranging from 0.8 to 1.25. 0.552 The bioequivalence of AUC 0.552 There were no significant differences between the two formulations with respect to asparaginase. Systemic exposure to asparaginase was similar between the two products, and repeated dosing resulted in some accumulation in both sexes. Asparagine was completely inhibited in all animals for up to 336 hours, and in the majority of animals for up to 552 hours.

[0243] In summary, there were no significant differences between the 500 IU / kg doses of either liquid pegaspargase or reconstituted lyophilized pegaspargase, and they had comparable pharmacokinetic, pharmacodynamic, and immunogenicity profiles.

[0244] [Embodiment] In one embodiment, the present disclosure provides a lyophilized, shelf-stable composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently attached to the asparaginase by a linker, the lyophilized, shelf-stable composition also comprising a buffer, a salt, and a sugar.

[0245] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 daltons.

[0246] In some embodiments, the asparaginase is E. coli asparaginase.

[0247] In some embodiments, the linker is a urethane linker. In some embodiments, the linker is a succinic acid linker.

[0248] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount ranging from 500 to 1,000 IU / g.

[0249] In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt.%. In some embodiments, the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt.%.

[0250] In some embodiments, the salt is sodium chloride. In some embodiments, sodium chloride is present in an amount ranging from 0.1 to 1 wt.%.

[0251] In some embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide comprises sucrose. In some embodiments, the sugar comprises sucrose in an amount ranging from 1 to 10 wt.%.

[0252] In some embodiments, the composition is present in a unit dose container. In some embodiments, the vial is a sealed glass vial.

[0253] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount ranging from 0.25 to 0.3 wt.% and sodium phosphate monobasic in an amount ranging from 0.05 to 0.07 wt.%, the salt being sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar being sucrose in an amount ranging from 4 to 5 wt.%.

[0254] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, and the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount of 0.279 wt.% and sodium phosphate monobasic in an amount of 0.06 wt.%, and the salt is sodium chloride in an amount of 0.425 wt.%.

[0255] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt.% and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt.%, the salt being sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar being sucrose in an amount ranging from 4 to 5 wt.%.

[0256] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount of 0.279 wt.% and sodium phosphate monobasic in an amount of 0.06 wt.%, the salt being sodium chloride in an amount of 0.425 wt.%, and the sugar being sucrose in an amount of 4.5 wt.%.

[0257] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, and the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount of 0.558 wt.% and sodium phosphate monobasic in an amount of 0.129 wt.%, and the salt is sodium chloride in an amount of 0.85 wt.%.

[0258] In another embodiment, the present disclosure provides a polyethylene glycol-asparaginase having a polyethylene glycol group covalently attached to E. coli asparaginase by a succinic acid linker. In another embodiment, the present disclosure provides a lyophilized, shelf-stable composition comprising asparaginase, the composition comprising polyethylene glycol-asparaginase, the polyethylene glycol group being covalently attached to E. coli asparaginase by a urethane linker. This lyophilized storage-stable composition may also include a phosphate buffer, a salt, and optionally a disaccharide as outlined in the embodiments above.

[0259] In another embodiment, the present disclosure provides a method of deaminating asparagine in a subject by administering a composition disclosed herein.

[0260] In some embodiments, the method comprises reconstituting a lyophilized storage-stable composition according to the present disclosure to produce a reconstituted dosage unit, and administering the reconstituted dosage unit to a subject to deaminate asparagine in the subject. In some embodiments, the reconstituting comprises mixing the lyophilized storage-stable composition with water for injection (WFI).

[0261] In some embodiments, the dosage unit contains 700-800 IU / mL of polyalkylene oxide-asparaginase.

[0262] In some embodiments, the dosage unit contains 2.5-6 mg / g dibasic sodium phosphate. In some embodiments, the dosage unit contains 2.5-3 mg / g dibasic sodium phosphate. In some embodiments, the dosage unit contains 5-6 mg / g dibasic sodium phosphate. In some embodiments, the dosage unit contains 5.25-5.75 mg / g dibasic sodium phosphate.

[0263] In some embodiments, the dosage unit contains 0.45-1.5 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 0.45-0.75 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 1-2 mg / g of monobasic sodium phosphate. In some embodiments, the dosage unit contains 1-1.5 mg / g of monobasic sodium phosphate.

[0264] In some embodiments, the dosage unit contains 4-9 mg / g sodium chloride. In some embodiments, the dosage unit contains 4-4.5 mg / g sodium chloride. In some embodiments, the dosage unit contains 8-9 mg / g sodium chloride.

[0265] In some embodiments, the dosage unit comprises sucrose, which in some embodiments is present in an amount ranging from 40 to 50 mg / g.

[0266] In some embodiments, the reconstituted dosage unit delivers between 1,500 and 3,000 IU / m2 of polyalkylene oxide-asparaginase to a subject. In some embodiments, the reconstituted dosage unit delivers between 2,000 and 2,750 IU / m2 of polyalkylene oxide-asparaginase to a subject.

[0267] In some embodiments, the method is a method of treating a neoplastic condition in a subject. In some embodiments, the neoplastic condition is cancer. In some embodiments, the cancer is leukemia. In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL). In some embodiments, the leukemia is acute myeloid leukemia (AML).

[0268] In some embodiments, the subject is prescribed a treatment regimen comprising an induction phase, a consolidation phase, and a maintenance phase. In some embodiments, the method comprises administering a single reconstituted dosage unit to the subject during the induction phase, and administering a plurality of reconstituted dosage units during the maintenance phase. In some embodiments, the plurality of reconstituted dosage units is administered to the subject by administering a reconstituted dosage unit to the subject every three weeks. In some embodiments, The multiple reconstituted dosage units are administered to the subject every two weeks. It is administered to the subject by

[0269] In some embodiments, the subject is a juvenile. In some embodiments, the subject is an adult.

[0270] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising dibasic sodium phosphate in an amount ranging from 2.5 to 3 mg / g and monobasic sodium phosphate in an amount ranging from 0.5 to 0.7 mg / g, the salt being sodium chloride in an amount ranging from 4 to 4.5 mg / g, and the sugar being sucrose in an amount ranging from 40 to 50 mg / g.

[0271] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount of 2.79 mg / g and sodium phosphate monobasic in an amount of 0.6 mg / g, the salt being sodium chloride in an amount of 4.25 mg / g, and the sugar being sucrose in an amount of 45 mg / g.

[0272] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising dibasic sodium phosphate in an amount ranging from 2.5 to 3 mg / g and monobasic sodium phosphate in an amount ranging from 0.5 to 0.7 mg / g, the salt being sodium chloride in an amount ranging from 4 to 4.5 mg / g, and the sugar being sucrose in an amount ranging from 40 to 50 mg / g.

[0273] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount of 2.79 mg / g and sodium phosphate monobasic in an amount of 0.6 mg / g, the salt being sodium chloride in an amount of 4.25 mg / g, and the sugar being sucrose in an amount of 45 mg / g.

[0274] In another embodiment, the present disclosure provides a method for producing a lyophilized polyalkylene oxide-asparaginase composition by lyophilizing an aqueous concentrated composition comprising a polyalkylene oxide-asparaginase having polyalkylene oxide groups covalently attached to the asparaginase by a linker, a buffer, a salt, and a sugar in a manner sufficient to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition.

[0275] In some embodiments, the concentrated aqueous composition comprises 1,500 to 3,000 IU / mL of polyalkylene oxide-asparaginase.

[0276] In some embodiments, the aqueous concentrate composition contains 0.1 to 0.5 wt.% dibasic phosphorus Contains sodium phosphate.

[0277] In some embodiments, the aqueous concentrate composition comprises 0.01 to 0.1 wt.% monobasic sodium phosphate.

[0278] In some embodiments, the aqueous concentrate composition comprises 0.1 to 1 wt.% sodium chloride. In some embodiments, the aqueous concentrate composition comprises sucrose.

[0279] In some embodiments, the sucrose is present in an amount ranging from 1 to 10 wt. %. In some embodiments, the method also includes a method for producing the aqueous concentrate composition.

[0280] In some embodiments, the method also includes introducing the aqueous concentrated composition into a unit dose container and lyophilizing the aqueous concentrated composition in the unit dose container. In some embodiments, the unit dose container is a vial. In some embodiments, the vial is a glass vial. In some embodiments, the method also includes sealing the lyophilized composition in the unit dose container.

[0281] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group comprises a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group comprises a molecular weight of 5,000 daltons.

[0282] In some embodiments, the asparaginase is E. coli asparaginase.

[0283] In some embodiments, the linker is a urethane linker. In some embodiments, the linker is a succinic acid linker.

[0284] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising sodium phosphate dibasic in an amount ranging from 0.25 to 0.3 wt.% and sodium phosphate monobasic in an amount ranging from 0.05 to 0.07 wt.%, the salt being sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar being sucrose in an amount ranging from 4 to 5 wt.%.

[0285] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %, the salt is sodium chloride in an amount ranging from 0.4 to 0.45 wt. %, and the sugar is sucrose in an amount ranging from 4 to 5 wt. %.

[0286] In other embodiments, the disclosure provides a kit comprising two or more unit dose containers each containing a lyophilized, shelf-stable composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently attached to the asparaginase by a linker, a buffer, a salt, and a sugar.

[0287] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 daltons.

[0288] In some embodiments, the asparaginase is E. coli asparaginase.

[0289] In some embodiments, the linker is a urethane linker. In some embodiments, the linker is a succinic acid linker.

[0290] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount ranging from 500 to 1,000 IU / g.

[0291] In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt.%. In some embodiments, the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt.%.

[0292] In some embodiments, the salt is sodium chloride. In some embodiments, sodium chloride is present in an amount ranging from 0.1 to 1 wt.%.

[0293] In some embodiments, the sugar is a disaccharide. In some embodiments, the disaccharide comprises sucrose. In some embodiments, the sugar comprises sucrose in an amount ranging from 1 to 10 wt.%.

[0294] In some embodiments, the unit dose container is a vial. In some embodiments, the unit dose container is a glass vial. In some embodiments, the vial is a sealed glass vial. In some embodiments, the unit dose container is sealed.

[0295] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount ranging from 0.25 to 0.3 wt.% and sodium phosphate monobasic in an amount ranging from 0.05 to 0.07 wt.%, the salt being sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar being sucrose in an amount ranging from 4 to 5 wt.%.

[0296] In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g, the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt.% and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt.%, the salt being sodium chloride in an amount ranging from 0.4 to 0.45 wt.%, and the sugar being sucrose in an amount ranging from 4 to 5 wt.%.

[0297] In another embodiment, the present disclosure includes a method of treating acute myeloid leukemia (AML) in a subject, the method comprising administering to the subject a dose of polyalkylene oxide-asparaginase effective to treat the acute myeloid leukemia (AML) in the subject, the polyalkylene oxide-asparaginase having a polyalkylene oxide covalently attached to the asparaginase by a linker.

[0298] In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group.

[0299] In some embodiments, the polyethylene glycol group comprises a molecular weight in the range of 2,000 to 10,000 daltons.

[0300] In some embodiments, the polyethylene glycol group comprises a molecular weight of 5,000 daltons.

[0301] In some embodiments, the asparaginase is E. coli asparaginase. In some embodiments, the linker is a urethane linker. In some embodiments, the linker is a succinic acid linker.

[0302] In some embodiments, the dose comprises an amount of polyalkylene oxide-asparaginase in the range of 700-800 IU / g.

[0303] In some embodiments, the dosage comprises a buffer and a salt. In some embodiments, the dosage comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dosage comprises 5.25-5.75 mg / g dibasic sodium phosphate. In some embodiments, the dosage comprises 1-1.5 mg / g monobasic sodium phosphate. In some embodiments, the salt is sodium chloride. In some embodiments, the dosage comprises 8-9 mg / g sodium chloride.

[0304] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is Escherichia coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising sodium phosphate dibasic in an amount ranging from 5.25 to 5.75 mg / g and sodium phosphate monobasic in an amount ranging from 1 to 1.5 mg / g, and the salt comprises sodium chloride in an amount ranging from 8 to 9 mg / g.

[0305] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising 5.58 mg / g of dibasic sodium phosphate and 1.29 mg / g of monobasic sodium phosphate, and the salt comprises 8.5 mg / g of sodium chloride.

[0306] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising 5.25-5.75 mg / g dibasic sodium phosphate and 1-1.5 mg / g monobasic sodium phosphate, and the salt comprises 8-9 mg / g sodium chloride.

[0307] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising 5.58 mg / g of dibasic sodium phosphate and 1.29 mg / g of monobasic sodium phosphate, and the salt comprises 8.5 mg / g of sodium chloride.

[0308] In some embodiments, the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate. In some embodiments, the dosage comprises 2.5-3 mg / g dibasic sodium phosphate. In some embodiments, the dosage comprises 0.45-0.75 mg / g monobasic sodium phosphate. In some embodiments, the salt is sodium chloride. In some embodiments, the dosage comprises 4-4.5 mg / g sodium chloride. In some embodiments, the disaccharide comprises sucrose. In some embodiments, sucrose is present in an amount ranging from 40-50 mg / g.

[0309] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising 2.5-3.5 mg of dibasic sodium phosphate and 0.5-0.7 mg of monobasic sodium phosphate, the salt being 4-4.5 mg / g of sodium chloride, and the sugar being 40-50 mg / g of sucrose.

[0310] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising 2.79 mg / g dibasic sodium phosphate and 0.6 mg / g monobasic sodium phosphate, the salt comprising 4.25 mg / g sodium chloride, and the sugar comprising 45 mg / g sucrose.

[0311] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising 2.5-3.5 mg of dibasic sodium phosphate and 0.5-0.7 mg of monobasic sodium phosphate, the salt being 4-4.5 mg / g of sodium chloride, and the sugar being 40-50 mg / g of sucrose.

[0312] In some embodiments, the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group having a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer is a phosphate buffer comprising 2.79 mg / g dibasic sodium phosphate and 0.6 mg / g monobasic sodium phosphate, the salt comprising 4.25 mg / g sodium chloride, and the sugar comprising 45 mg / g sucrose.

[0313] In some embodiments, the method also includes creating a dose by reconstituting a lyophilized storage-stable composition.

[0314] In some embodiments, the dose is 1,500 to 3,000 IU / m 2 Polyal The dose of 2,000 to 2,750 IU / m is delivered to the subject. 2 of polyalkylene oxide-asparaginase is delivered to the subject.

[0315] In some embodiments, the subject is prescribed a treatment regimen comprising an induction phase, a consolidation phase, and a maintenance phase. In some embodiments, the method comprises administering a single reconstituted dosage unit to the subject during the induction phase and administering multiple reconstituted dosage units during the maintenance phase. In some embodiments, the multiple reconstituted dosage units are administered to the subject by administering a reconstituted dosage unit to the subject every three weeks. In some embodiments, the multiple reconstituted dosage units are administered to the subject by administering a reconstituted dosage unit to the subject every two weeks.

[0316] In some embodiments, the subject is a juvenile. In some embodiments, the subject is an adult.

[0317] While the foregoing embodiments have been described by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art, in view of the teachings of this disclosure, that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

[0318] Thus, the foregoing merely illustrates the principles of the embodiments of the present disclosure. It will be understood that, although not explicitly described or shown herein, those skilled in the art will be able to devise various arrangements that embody the principles of the embodiments of the present disclosure and are within the spirit and scope of the present invention. Furthermore, all examples and conditional statements recited herein are intended primarily to aid the reader in understanding the principles of the embodiments of the present disclosure and the concepts that the inventors have contributed to expanding the art, and should be construed as not including limitations on such specifically recited examples and conditions. Furthermore, all statements herein reciting principles, aspects, and embodiments of the present disclosure are intended to encompass both structural and functional equivalents thereof, as well as specific examples thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the embodiments of the present disclosure, therefore, is not intended to be limited to the embodiments shown and described herein. Rather, the scope and spirit of the embodiments of the present disclosure are embodied by the appended claims.

Claims

1. 1. A lyophilized, storage-stable composition comprising: a polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently attached to the asparaginase by a linker; Buffer; Salt and sugar A composition comprising:

2. 2. The lyophilized, storage-stable composition of claim 1, wherein the polyalkylene oxide group comprises a polyethylene glycol group.

3. 3. The lyophilized, storage-stable composition of claim 2, wherein the polyethylene glycol group has a molecular weight of 2,000 to 10,000 daltons.

4. 4. The lyophilized, storage-stable composition of claim 3, wherein the polyethylene glycol group has a molecular weight of 5,000 daltons.

5. The freeze-dried, shelf-stable composition of any of claims 1 to 4, wherein the asparaginase is E. coli asparaginase.

6. 10. The lyophilized storage-stable composition of any of the above claims, wherein the linker is a urethane linker.

7. 10. The lyophilized storage-stable composition of any of the above claims, wherein the linker is a succinic acid linker.

8. 10. The lyophilized, storage-stable composition of any of the above claims, wherein the polyalkylene oxide-asparaginase is present in an amount ranging from 500 to 1,000 IU / g.

9. 10. The lyophilized storage-stable composition of any of the above claims, wherein the buffer comprises a phosphate buffer.

10. 10. The lyophilized, storage-stable composition of claim 9, wherein the phosphate buffer comprises sodium phosphate dibasic and sodium phosphate monobasic.

11. 11. The lyophilized, storage-stable composition of claim 10, wherein the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt. %.

12. 12. The lyophilized, storage-stable composition of claim 10 or 11, wherein the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt. %.

13. 10. The freeze-dried storage-stable composition of any of the above claims, wherein the salt is sodium chloride.

14. 14. The lyophilized, storage-stable composition of claim 13, wherein the sodium chloride is present in an amount ranging from 0.1 to 1 wt. %.

15. 10. The freeze-dried, shelf-stable composition of any of the preceding claims, wherein the sugar comprises a disaccharide. thing.

16. 16. The freeze-dried, shelf-stable composition of claim 15, wherein the disaccharide comprises sucrose.

17. 17. The freeze-dried, shelf-stable composition of claim 15 or 16, wherein the sugar comprises sucrose in an amount ranging from 1 to 10 wt.%.

18. 10. The lyophilized, storage-stable composition of any of the above claims, wherein the composition is present in a unit dose container.

19. 20. The lyophilized storage-stable composition of claim 18, wherein the unit dose container is a vial.

20. 20. The lyophilized storage-stable composition of claim 19, wherein the vial is a sealed glass vial.

21. 21. A freeze-dried, storage-stable composition according to any one of claims 1 to 5, or 8 to 20, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a urethane linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and The sugars include sucrose in an amount ranging from 4 to 5 wt. %. A freeze-dried, storage-stable composition.

22. 21. A freeze-dried, storage-stable composition according to any one of claims 1 to 5, or 8 to 20, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a succinic acid linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and The sugars include sucrose in an amount ranging from 4 to 5 wt. %. A freeze-dried, storage-stable composition.

23. 1. A lyophilized, storage-stable composition comprising: a polyalkylene oxide-asparaginase comprising a polyethylene glycol group covalently attached to the asparaginase by a urethane linker; phosphate buffer; Salt and disaccharide A composition comprising:

24. 1. A lyophilized, storage-stable composition comprising: polyethylene glycol-asparaginase, which comprises a polyethylene glycol group covalently attached to E. coli asparaginase by a succinic acid linker; phosphate buffer; salt; and disaccharide 1. A freeze-dried, storage-stable composition comprising:

25. 25. The lyophilized storage-stable composition of claim 23 or 24, wherein the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons.

26. 26. The lyophilized storage-stable composition of claim 25, wherein the polyethylene glycol group has a molecular weight of 5,000 daltons.

27. 27. The lyophilized, storage-stable composition of any of claims 23-26, wherein the polyethylene glycol-asparaginase is present in an amount ranging from 500 to 1,000 IU / g.

28. 28. The lyophilized storage-stable composition of any of claims 23-27, wherein the phosphate buffer comprises dibasic sodium phosphate and monobasic sodium phosphate.

29. 29. The lyophilized, storage-stable composition of claim 28, wherein the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.5 wt.%.

30. 30. The lyophilized, storage-stable composition of claim 28 or 29, wherein the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.1 wt.%.

31. 31. The lyophilized storage-stable composition of any of claims 23 to 30, wherein the salt is sodium chloride.

32. 32. The lyophilized, storage-stable composition of claim 31, wherein the sodium chloride is present in an amount ranging from 0.1 to 1 wt. %.

33. 33. The freeze-dried storage-stable composition of any of claims 23-32, wherein the disaccharide comprises sucrose.

34. 34. The lyophilized, shelf-stable composition of claim 33, wherein the disaccharide comprises sucrose in an amount ranging from 1 to 10 wt.%.

35. 35. The lyophilized, storage-stable composition of any of claims 23-34, wherein the composition is present in a unit dose container.

36. 36. The lyophilized storage-stable composition of claim 35, wherein the unit dose container is a vial.

37. 37. The lyophilized storage-stable composition of claim 36, wherein the vial is a sealed glass vial.

38. 1. A lyophilized, storage-stable composition comprising: i) polyethylene glycol-asparaginase, which comprises polyethylene glycol covalently bonded to E. coli asparaginase by a urethane linker, The alkylene oxide-asparaginase is present in an amount of 750 IU / g, and the polyethylene The ethylene glycol group has a molecular weight of 5,000 daltons; ii) a buffer solution comprising a phosphate buffer solution containing 0.279 wt.% dibasic sodium phosphate and 0.06 wt.% monobasic sodium phosphate; iii) 0.425 wt. % sodium chloride; and iv) sucrose in an amount of 4.5 wt. % 1. A freeze-dried, storage-stable composition comprising:

39. 1. A lyophilized, storage-stable composition comprising: i) polyethylene glycol-asparaginase, which comprises polyethylene glycol covalently bound to E. coli asparaginase by a succinic acid linker, The alkylene oxide-asparaginase is present in an amount of 750 IU / g, and the polyethylene The ethylene glycol group has a molecular weight of 5,000 daltons; ii) a buffer solution comprising a phosphate buffer solution containing 0.279 wt.% dibasic sodium phosphate and 0.06 wt.% monobasic sodium phosphate; iii) 0.425 wt. % sodium chloride; and iv) sucrose in an amount of 4.5 wt. % 1. A freeze-dried, storage-stable composition comprising:

40. 1. A liquid composition comprising: i) polyethylene glycol-asparaginase, which comprises polyethylene glycol covalently bonded to E. coli asparaginase by a urethane linker, The alkylene oxide-asparaginase is present in an amount of 750 IU / g, and the polyethylene The ethylene glycol group has a molecular weight of 5,000 daltons; ii) a buffer solution comprising a phosphate buffer solution containing 0.558 wt. % dibasic sodium phosphate and 0.129 wt. % monobasic sodium phosphate; and iii) 0.85 wt. % sodium chloride; A liquid composition comprising:

41. 41. A method of deaminating asparagine in a subject, comprising: administering to the subject the composition of claim 40 in dosage unit form to deaminate asparagine in the subject.

42. 1. A method for deaminating asparagine in a subject, comprising: Reconstituting a lyophilized storage-stable composition according to any one of claims 1 to 39 to produce a reconstituted dosage unit; and administering said reconstituted dosage unit to a subject to deaminate asparagine in said subject. A method comprising:

43. 43. The method of claim 42, wherein said reconstituting comprises mixing said lyophilized storage-stable composition with water for injection (WFI).

44. 44. The method of any one of claims 41 to 43, wherein the dosage unit comprises 700 to 800 IU / mL of polyalkylene oxide-asparaginase.

45. 45. The method of any of claims 41 to 44, wherein the dosage unit contains 2.5 to 6 mg / g of dibasic sodium phosphate.

46. 46. ​​The method of any of claims 41 to 45, wherein the dosage unit comprises 0.45 to 1.5 mg / g of monobasic sodium phosphate.

47. 47. The method of any of claims 41 to 46, wherein the dosage unit contains 4 to 9 mg / g sodium chloride.

48. 48. The method of any one of claims 41 to 47, wherein the dosage unit comprises sucrose.

49. 49. The method of claim 48, wherein the sucrose is present in an amount ranging from 40 to 50 mg / g.

50. 1,500-3,000IU / m ２ 50. The method of any of claims 41 to 49, wherein a polyalkylene oxide-asparaginase of the formula:

51. 2,000-2,750IU / m ２ 51. The method of claim 50, wherein a polyalkylene oxide-asparaginase of

52. 52. The method of any of claims 41 to 51, which is a method for treating a neoplastic condition in said subject.

53. 53. The method of claim 52, wherein the neoplastic condition is cancer.

54. 54. The method of claim 53, wherein the neoplastic condition is leukemia.

55. 55. The method of claim 54, wherein the leukemia is acute lymphoblastic leukemia (ALL).

56. 55. The method of claim 54, wherein the leukemia is acute myeloid leukemia (AML).

57. 57. The method of any of claims 52-56, wherein the subject is prescribed a treatment regimen comprising an induction phase, a consolidation phase, and a maintenance phase.

58. 58. The method of claim 57, comprising administering a single dosage unit to the subject in the induction phase and administering multiple dosage units during the maintenance phase.

59. 59. The method of claim 58, comprising administering a plurality of dosage units to the subject by administering a dosage unit to the subject every three weeks.

60. 59. The method of claim 58, comprising administering a plurality of dosage units to the subject by administering a dosage unit to the subject every two weeks.

61. The method of any one of claims 41 to 60, wherein the subject is a minor.

62. The method of any one of claims 41 to 60, wherein the subject is an adult.

63. 63. The method of any one of claims 41 to 62, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / mL; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is E. coli asparaginase; the linker is a urethane linker; The buffer contains sodium phosphate dibasic in an amount ranging from 2.5 to 3 mg / g, and 0.5 a phosphate buffer containing monobasic sodium phosphate in an amount ranging from 0.7 mg / g to 0.7 mg / g; The salt comprises sodium chloride in an amount ranging from 4 to 4.5 mg / g; and The sugars include sucrose in an amount ranging from 40 to 50 mg / g. A freeze-dried, storage-stable composition.

64. 63. The method of any one of claims 41 to 62, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / mL; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a succinic acid linker; and the buffer comprises a phosphate buffer containing dibasic sodium phosphate in an amount ranging from 2.5 to 3 mg / g and monobasic sodium phosphate in an amount ranging from 0.5 to 0.7 mg / g; The salt comprises sodium chloride in an amount ranging from 4 to 4.5 mg / g; and The sugars include sucrose in an amount ranging from 40 to 50 mg / g. A freeze-dried, storage-stable composition.

65. the dibasic sodium phosphate in an amount of 2.79 mg / g; the monobasic sodium phosphate in an amount of 0.6 mg / g; the sodium chloride in an amount of 4.25 mg / g; and 65. The method of claim 63 or 64, wherein the sucrose is in an amount of 45 mg / g.

66. 1. A method of making a lyophilized polyalkylene oxide-asparaginase composition, comprising: (i) a polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently attached to an asparaginase by a linker; (ii) buffer; (iii) a salt; and (iii) sugar; lyophilizing an aqueous concentrated composition comprising:

67. 67. The method of claim 66, wherein the concentrated aqueous composition comprises 1,500 to 3,000 IU / mL of polyalkylene oxide-asparaginase.

68. 68. The method of claim 66 or 67, wherein the aqueous concentrate composition comprises 0.1 to 0.5 wt.% dibasic sodium phosphate.

69. 69. The method of any of claims 66-68, wherein the aqueous concentrate composition comprises 0.01 to 0.1 wt.% monobasic sodium phosphate.

70. 70. The method of any of claims 66 to 69, wherein the aqueous concentrate composition comprises 0.1 to 1 wt.% sodium chloride.

71. 71. The method of any of claims 66-70, wherein the aqueous concentrated composition comprises sucrose.

72. 72. The method of claim 71, wherein the sucrose is present in an amount ranging from 1 to 10 wt.%.

73. 73. The method of any of claims 66-72, wherein said method further comprises producing said aqueous concentrate composition.

74. 74. The method of any of claims 66-73, further comprising introducing the aqueous concentrate composition into a unit dose container and lyophilizing the aqueous concentrate composition in the unit dose container.

75. 75. The method of claim 74, wherein the unit dose container is a vial.

76. 76. The method of claim 75, wherein the unit dose container is a glass vial.

77. 77. The method of any of claims 74-76, further comprising sealing the lyophilized composition in the unit dose container.

78. 78. The method of any one of claims 66 to 77, wherein the polyalkylene oxide group comprises a polyethylene glycol group.

79. 79. The method of claim 78, wherein the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons.

80. 80. The method of claim 79, wherein the polyethylene glycol group has a molecular weight of 5,000 daltons.

81. 81. The method of any one of claims 66 to 80, wherein the asparaginase is Escherichia coli asparaginase.

82. The method of any one of claims 66 to 81, wherein the linker is a urethane linker.

83. The method of any one of claims 66 to 82, wherein the linker is a succinic acid linker.

84. 82. The method of any one of claims 66 to 81, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a urethane linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and the sugar comprises sucrose in an amount ranging from 4 to 5 wt. %.

85. 82. The method according to any one of claims 66 to 81, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a succinic acid linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and the sugar comprises sucrose in an amount ranging from 4 to 5 wt. %.

86. 1. A method of treating acute myeloid leukemia (AML) in a subject, comprising:

1. A method comprising administering to a subject a dose of polyalkylene oxide-asparaginase in an amount effective to treat AML in the subject, wherein the polyalkylene oxide-asparaginase comprises a polyalkylene oxide group covalently attached to the asparaginase by a linker.

87. 87. The method of claim 86, wherein the polyalkylene oxide group comprises a polyethylene glycol group.

88. 88. The method of claim 87, wherein the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons.

89. 89. The method of claim 88, wherein the polyethylene glycol group has a molecular weight of 5,000 daltons.

90. 90. The method of any one of claims 86 to 89, wherein the asparaginase is Escherichia coli asparaginase.

91. The method of any one of claims 86 to 90, wherein the linker is a urethane linker.

92. The method of any one of claims 86 to 90, wherein the linker is a succinic acid linker.

93. 93. The method of any of claims 86-92, wherein the dose comprises 700-800 IU / mL of the polyalkylene oxide-asparaginase.

94. 94. The method of any of claims 86-93, wherein the dose further comprises a buffer and a salt.

95. 95. The method of claim 94, wherein the buffer comprises a phosphate buffer.

96. 96. The method of claim 95, wherein the phosphate buffer comprises sodium phosphate dibasic and sodium phosphate monobasic.

97. 97. The method of claim 96, wherein the dosage comprises 5.25 to 5.75 mg / g of dibasic sodium phosphate.

98. 98. The method of any of claims 95-97, wherein the dose comprises 1-1.5 mg / g of monobasic sodium phosphate.

99. 99. The method of any one of claims 86 to 98, wherein the salt is sodium chloride.

100. 100. The method of claim 99, wherein the dose comprises 8-9 mg / g sodium chloride.

101. 87. The method of claim 86, wherein the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising 5.25-5.75 mg / g dibasic sodium phosphate and 1-1.5 mg / g monobasic sodium phosphate, and the salt comprises 8-9 mg / g sodium chloride.

102. 102. The method of claim 101, wherein the dose comprises a phosphate buffer comprising 5.58 mg / g dibasic sodium phosphate and 1.29 mg / g monobasic sodium phosphate, and the salt comprises 8.5 mg / g sodium chloride.

103. 87. The method of claim 86, wherein the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising 5.25-5.75 mg / g dibasic sodium phosphate and 1-1.5 mg / g monobasic sodium phosphate, and the salt comprises 8-9 mg / g sodium chloride.

104. 104. The method of claim 103, wherein the dose comprises a phosphate buffer comprising 5.58 mg / g dibasic sodium phosphate and 1.29 mg / g monobasic sodium phosphate, and the salt comprises 8.5 mg / g sodium chloride.

105. 94. The method of any of claims 86-93, wherein the dose further comprises a buffer, a salt and a sugar.

106. 106. The method of claim 105, wherein the buffer comprises a phosphate buffer.

107. 107. The method of claim 106, wherein the phosphate buffer comprises sodium phosphate dibasic and sodium phosphate monobasic.

108. 108. The method of claim 107, wherein the dose comprises 2.5 to 3 mg / g of dibasic sodium phosphate.

109. 109. The method of claim 107 or 108, wherein the dose comprises 0.45 to 0.75 mg / g of monobasic sodium phosphate.

110. 110. The method of any one of claims 105 to 109, wherein the salt is sodium chloride.

111. 111. The method of claim 110, wherein the dose comprises 4 to 4.5 mg / g of salt.

112. 112. The method of any one of claims 105 to 111, wherein the sugar comprises a disaccharide.

113. 113. The method of claim 112, wherein the disaccharide comprises sucrose.

114. 114. The method of claim 113, wherein the sucrose is present in an amount ranging from 40 to 50 mg / g.

115. 106. The method of claim 105, wherein the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a urethane linker, the buffer comprises a phosphate buffer comprising 2.5-3.0 mg / g dibasic sodium phosphate and 0.5-0.7 mg / g monobasic sodium phosphate, and the salt comprises 4-4.5 mg / g sodium chloride, and the sugar comprises 40-50 mg / g sucrose.

116. 116. The method of claim 115, wherein the dose comprises a phosphate buffer comprising 2.79 mg / g dibasic sodium phosphate and 0.6 mg / g monobasic sodium phosphate, the salt comprising sodium chloride in an amount of 4.25 mg / g, and the sugar comprising sucrose in an amount of 45 mg / g.

117. 106. The method of claim 105, wherein the dose comprises 750 IU / g of polyalkylene oxide-asparaginase, the polyethylene glycol group has a molecular weight of 5,000 daltons, the asparaginase is E. coli asparaginase, the linker is a succinic acid linker, the buffer comprises a phosphate buffer comprising 2.5-3.0 mg / g dibasic sodium phosphate and 0.5-0.7 mg / g monobasic sodium phosphate, and the salt comprises 4-4.5 mg / g sodium chloride, and the sugar comprises 40-50 mg / g sucrose.

118. 118. The method of claim 117, wherein the dose comprises a phosphate buffer comprising 2.79 mg / g dibasic sodium phosphate and 0.6 mg / g monobasic sodium phosphate, the salt comprising sodium chloride in an amount of 4.25 mg / g, and the sugar comprising sucrose in an amount of 45 mg / g.

119. 119. The method of any of claims 105-118, further comprising preparing said dose by reconstituting a lyophilized storage-stable composition.

120. The dose is 1,500 to 3,000 IU / m ２ 120. The method of any of claims 86-119, wherein a polyalkylene oxide-asparaginase of the formula:

121. The dose is 2,000 to 2,750 IU / m ２ 121. The method of claim 120, wherein a polyalkylene oxide-asparaginase of

122. 122. The method of any of claims 86-121, wherein the subject is prescribed a treatment regimen comprising an induction phase, a consolidation phase, and a maintenance phase.

123. 123. The method of claim 122, comprising administering a single dosage unit to the subject during the induction phase and administering multiple dosage units during the maintenance phase.

124. 124. The method of claim 123, comprising administering a plurality of dosage units to the subject by administering a dosage unit to the subject every three weeks.

125. 124. The method of claim 123, comprising administering a plurality of dosage units to the subject by administering a dosage unit to the subject every two weeks.

126. 126. The method of any one of claims 86 to 125, wherein the subject is a minor.

127. The method of any one of claims 86 to 125, wherein the subject is an adult.

128. polyalkylene oxide-asparaginases, each comprising a polyalkylene oxide group covalently attached to an asparaginase by a linker; a buffer solution; and salt A kit comprising one or more unit dose containers containing a composition comprising:

129. polyalkylene oxide-asparaginases, each comprising a polyalkylene oxide group covalently attached to an asparaginase by a linker; Buffer; salt; and sugar A kit comprising one or more unit dose containers containing a lyophilized storage-stable composition comprising:

130. 130. The kit of claim 128 or 129, wherein the polyalkylene oxide group comprises a polyethylene glycol group.

131. 131. The kit of claim 130, wherein the polyethylene glycol group has a molecular weight in the range of 2,000 to 10,000 daltons.

132. 132. The kit of claim 131, wherein the polyethylene glycol group has a molecular weight of 5,000 daltons.

133. 133. The kit of any one of claims 128 to 132, wherein the asparaginase is Escherichia coli asparaginase.

134. The kit of any one of claims 128 to 133, wherein the linker is a urethane linker.

135. The kit of any one of claims 128 to 133, wherein the linker is a succinic acid linker.

136. 134. The kit of any of claims 128-133, comprising: the polyalkylene oxide-asparaginase is present in an amount ranging from 500 to 1,000 IU / g.

137. 137. The kit of any of claims 128-136, wherein the buffer comprises a phosphate buffer.

138. 138. The kit of claim 137, wherein the phosphate buffer comprises sodium phosphate dibasic and sodium phosphate monobasic.

139. 139. The kit of claim 138, wherein the dibasic sodium phosphate is present in an amount ranging from 0.1 to 0.6 wt.%.

140. 140. The kit of claim 138 or 139, wherein the monobasic sodium phosphate is present in an amount ranging from 0.01 to 0.2 wt.%.

141. 141. The kit of any one of claims 128 to 140, wherein the salt is sodium chloride.

142. 142. The kit of claim 141, wherein sodium chloride is present in an amount ranging from 0.1 to 1 wt. %.

143. 143. The kit of any of claims 128-142, wherein the sugar comprises a disaccharide.

144. 144. The kit of claim 143, wherein the disaccharide comprises sucrose.

145. 145. The kit of claim 143 or 144, wherein the sugar comprises an amount in the range of 1 to 10 wt.% of sucrose.

146. 146. The kit of any of claims 128-145, wherein the unit dose container is a vial.

147. 147. The kit of claim 146, wherein the unit dose container is a glass vial.

148. 148. The kit of any of claims 128-147, wherein the unit dose container is hermetically sealed.

149. 149. The kit of any one of claims 129-133 or 136-148, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a urethane linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and The sugar comprises sucrose in an amount ranging from 4 to 5 wt. %.

150. 149. The kit of claims 129-133 or 136-148, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a succinic acid linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 0.25 to 0.3 wt. % and monobasic sodium phosphate in an amount ranging from 0.05 to 0.07 wt. %; The salt comprises sodium chloride in an amount ranging from 0.4 to 0.45 wt. %; and The sugar comprises sucrose in an amount ranging from 4 to 5 wt. %.

151. 149. The kit of any of claims 128-133 or 136-148, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a urethane linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 5.25 to 6.0 wt. % and monobasic sodium phosphate in an amount ranging from 1.0 to 1.75 wt. %; The salt comprises sodium chloride in an amount ranging from 8.0 to 9.0 wt. %.

152. 149. The kit of any of claims 128-133 or 136-148, comprising: the polyalkylene oxide-asparaginase is present in an amount of 750 IU / g; the polyalkylene oxide group comprises a polyethylene glycol group having a molecular weight of 5,000 daltons; the asparaginase is Escherichia coli asparaginase; the linker is a succinic acid linker; The buffer solution comprises a phosphate buffer solution containing dibasic sodium phosphate in an amount ranging from 5.25 to 6.0 wt. % and monobasic sodium phosphate in an amount ranging from 1.0 to 1.75 wt. %; The salt comprises sodium chloride in an amount ranging from 8.0 to 9.0 wt. %.

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