HK40007007B - Formulations of polyalkylene oxide-asparaginase and methods of making and using the same - Google Patents

Description

Polyalkylene oxide-asparaginase preparation and preparation and use method thereof

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims the benefit of 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 herein by reference in its entirety.

Preface

L-asparaginase is an enzyme that hydrolyzes to produce L-aspartic acid and ammonia through a deamination reaction. Escherichia coli (E. coli) contains two asparaginase isozymes: L-asparaginase I and L-asparaginase II. L-asparaginase I is located in the cytosol and can have an affinity for asparagine. However, L-asparaginase II is located in the cytosol and has a high affinity for L-asparagine. E. coli L-asparaginase II is a tetramer of identical subunits. E. coli L-asparaginase II is also known as L-asparagine amidohydrolase EC-2 type EC3.5.1.1.

Known L-asparaginase has therapeutic value in the treatment of leukemia. L-asparaginase is an amidohydrolase that catalyzes L-asparagine into L-aspartic acid and ammonia. It plays a major role in the metabolism of L-asparagine in plants, animals and microorganisms. What is fully established at present is that the therapeutic activity of this enzyme is caused by the disappearance/removal of circulating L-asparagine, which is an essential nutrient for tumor (leukemia) cell proliferation and survival, and the synthetic capacity of the L-asparagine of tumor cells is impaired, but not impaired for normal cells. Use L-asparaginase to induce tumor cell selective death to leukemia patients by hydrolyzing L-asparagine, resulting in the treatment of malignant tumors.

In some cases, L-asparaginase itself has the drawbacks of typical protein therapeutics, such as a high rate of clearance of foreign proteins from the patient and the potential to induce an immune response in patients treated with the enzyme. To address these drawbacks, polyethylene glycol-conjugated derivatives of L-asparaginase (PEG-asparaginase) can be used. PEG-asparaginases can be produced using L-asparaginase II extracted from Escherichia coli, and they can be substantially non-antigenic and can show reduced clearance from the patient's circulation.

PEG-asparaginase liquid injection formulation has been previously approved for commercial sale by the U.S. Food and Drug Administration. It is approved as a first-line treatment for patients with acute lymphoblastic leukemia (ALL) as a component of a multi-active agent chemotherapy regimen. In addition, it is approved for the treatment of patients with ALL and hypersensitivity to asparaginase (e.g., the native form of L-asparaginase).

Overview

Aspects of the present invention include polyalkylene oxide-asparaginase compositions. In some cases, the compositions include one or more of a buffer and a salt. In other aspects, the compositions are freeze-dried, storage-stable compositions. In some cases, the freeze-dried compositions include one or more of a buffer, a salt, and a sugar. Aspects of the present invention also include methods for preparing the compositions. These compositions are used in various applications, such as treating a tumor condition in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a process flow diagram for preparing a lyophilized storage-stable composition according to an embodiment of the present disclosure.

2 is a graph showing the purity (%) versus time (weeks) at 40° C. for a lyophilized, storage-stable composition according to an embodiment of the present disclosure.

3 shows a graphical representation of the potency (IU/mL) versus time (weeks) at 40° C. for lyophilized, storage-stable compositions according to embodiments of the present disclosure.

4 is a graph showing the relationship between purity (%) and time (weeks) at 25° C. for a lyophilized, storage-stable composition according to an embodiment of the present disclosure.

5 shows a graphical representation of potency (IU/mL) versus time (weeks) at 25° C. for lyophilized, storage-stable compositions according to embodiments of the present disclosure.

Figure 6 shows a process flow diagram for preparing a lyophilized, storage-stable composition according to an embodiment of the present disclosure. The final formulation and filtration steps are shown.

Figure 7 shows a process flow diagram for preparing a lyophilized, storage-stable composition according to an embodiment of the present disclosure. Aseptic filling and lyophilization steps are shown.

FIG8 is a graph showing the relationship between GF-HPLC (%) and time (months) at 2-8° C. (eg, 5° C.) for a lyophilized composition according to an embodiment of the present disclosure.

9 is a graph showing the relationship between potency (activity) (IU/mL) and time (months) at 2-8° C. (eg, 5° C.) of a lyophilized composition according to an embodiment of the present disclosure.

FIG10 is a graph showing the relationship between total aggregates and time (months) as measured by GF-HPLC at 2-8° C. (eg, 5° C.) for a lyophilized composition according to an embodiment of the present disclosure.

11 is a graph showing the relationship between the purity (%) and time (months) as determined by GF-HPLC for a lyophilized composition stored under accelerated conditions (25±3° C.; 60%±5% RH) according to an embodiment of the present disclosure.

12 is a graph showing the potency (activity) (IU/mL) versus time (months) of a lyophilized composition stored under accelerated conditions (25±3° C.; 60%±5% RH) according to an embodiment of the present disclosure.

13 is a graph showing the relationship between total aggregates as measured by GF-HPLC and time (months) for lyophilized compositions stored under accelerated conditions (25±3° C.; 60%±5% RH) according to an embodiment of the present disclosure.

14 is a graph showing the relationship between the purity (%) measured by GF-HPLC and time (months) of a lyophilized composition stored under heat load conditions (40±2° C.; 75%±5% RH) according to an embodiment of the present disclosure.

15 is a graph showing the relationship between potency (activity) (IU/mL) and time (months) for lyophilized compositions stored under heat load conditions (40±2° C.; 75%±5% RH) according to an embodiment of the present disclosure.

16 is a graph showing the relationship between total aggregates measured by GF-HPLC and time (months) for lyophilized cells stored under heat load conditions (40±2° C.; 75%±5% RH) according to an embodiment of the present disclosure.

definition

In describing embodiments of the present disclosure, the following terminology may be employed and is intended to be defined as follows.

By "substantially purified" is meant an isolated substance such that the substance comprises a majority of the sample in which it resides. For example, a substantially purified sample comprises 50% or more of the substance of interest, such as 60% or more of the substance of interest, such as 75% or more of the substance of interest, such as 90% or more of the substance of interest, such as 95% or more of the substance of interest, including 99% or more of the substance of interest. Any convenient protocol may be used to purify the substance of interest, including, but not limited to, filtration (e.g., diafiltration, ultrafiltration, etc.), selective precipitation, crystallization, ion exchange chromatography, affinity chromatography, and density-based precipitation.

By "isolated" is meant to describe a compound of interest that is different from the environment in which the compound naturally occurs. "Isolated" is meant to include a sample in which the compound of interest is enriched and/or in which the compound of interest is partially or substantially purified.

The terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administering the compositions of the present disclosure, and include humans and non-human animals. Examples of subjects include, but are not limited to, humans, chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds including domestic, wild, and game birds such as chickens, turkeys and other quail, ducks, geese, and the like. The terms do not denote a particular age. Thus, adults, adolescents, and newborn individuals are of interest.

"Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound or composition sufficient to treat a particular disorder or disease or one or more of its symptoms and/or to prevent the onset of the disease or disorder. With respect to oncological conditions, a pharmaceutically or therapeutically effective amount includes an amount sufficient to cause a reduction in and/or development of cancer and/or reduce the growth rate of cancer in a subject.

As used herein, the term "treat" refers to treating a disease or medical condition in a patient, such as a mammal (e.g., a human), including: (a) preventing the disease or medical condition from occurring, such as by treating a subject prophylactically; (b) ameliorating the disease or medical condition, such as by eliminating the disease or medical condition in a patient or causing its regression; (c) inhibiting the disease or medical condition, such as by slowing or arresting the progression of the disease or medical condition in a patient; or (d) relieving the symptoms of the disease or medical condition in a patient.

The term "physiological conditions" is meant to encompass those conditions that are compatible with living cells, such as primarily aqueous conditions of temperature, pH, salinity, etc. that are compatible with living cells.

Before describing the embodiments of the present disclosure in more detail, it should be understood that the embodiments are not limited to the specific embodiments described herein; as such, the embodiments may vary. It should also be understood that the terminology used herein is not intended to be limiting and is merely used to describe specific embodiments. 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. If a numerical range is provided, it should be understood that, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range and any other stated or intervening value in that range, to one-tenth of the unit of the lower limit, is included in the embodiments of the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the embodiments of the present disclosure, subject to any specifically excluded limits in the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present disclosure. The term "about" is used herein to provide literal support for the exact number that precedes it, as well as for numbers that are close to or approximately the number that precedes it. In determining whether a value is near or approximately a specifically recited value, a near or approximate unrecited value may be a value that, in the context in which it is presented, provides a substantial equivalent to the specifically recited value.

All publications, patents, and patent applications cited in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In addition, each cited publication, patent, or patent application is incorporated herein by reference to disclose and describe the subject matter related to the cited publication. The citation of any publication is for its disclosure prior to its filing date and should not be construed as an admission that the inventions described herein are not entitled to antedate such publications by virtue of prior invention. In addition, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

It should be noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a premise basis for the use of proprietary terms such as "alone", "only", etc., in connection with the description of "claim" elements or the application of "negative" limitations. As will be apparent to those skilled in the art when reading this disclosure, the various embodiments described and illustrated herein each have independent elements and features, which are easy to be separated from or combined with any other embodiments without departing from the scope or spirit of the embodiments of the present disclosure. Any enumerated method can be performed in the order of the events described or in any other logically possible order. Although the same methods and materials as those described herein can also be used to implement or test embodiments of the present disclosure, representative exemplary methods and materials are now described.

Detailed Description of the Invention

Aspects of the present invention include polyalkylene oxide-asparaginase compositions. In some cases, the compositions include one or more of a buffer and a salt. Aspects of the present invention also include methods of preparing the compositions. These compositions are useful in various applications, such as treating a tumor condition in a subject.

Aspects of the present invention include freeze-dried, storage-stable polyalkylene oxide-asparaginase compositions. In some cases, the freeze-dried compositions include one or more of a buffer, a salt, and a sugar. Aspects of the present invention also include methods for preparing the compositions. These compositions are used in various applications, such as treating a neoplastic condition (e.g., acute myeloid leukemia (AML)) in a subject.

Aspects of the present invention include methods for treating AML in a subject. The methods include administering to the subject a polyalkylene oxide-asparaginase in an amount effective to treat the subject's AML. Aspects of the present invention also include compositions comprising the polyalkylene oxide-asparaginase and kits useful in the subject methods.

In further describing embodiments of the present disclosure, compositions (eg, liquid and lyophilized) are first described in more detail. Next, methods of making, methods of using, and kits comprising the subject compositions are also described.

Composition

Aspects of the present disclosure include compositions of polyalkylene oxide-asparaginase comprising a polyalkylene oxide group covalently linked to asparaginase via a linker. The composition may further comprise one or more of a buffer and a salt. In certain embodiments, the composition is a freeze-dried, storage-stable composition. The freeze-dried, storage-stable composition may further comprise one or more of a buffer, a salt, and a sugar.

As described herein, the compositions of the present disclosure can include polyalkylene oxide-asparaginase. Polyalkylene oxide-asparaginase includes asparaginase covalently linked to one or more polyalkylene oxide groups via a linker. Asparaginase is an enzyme that can be composed of four identical subunits, each tetramer having one active site. For example, asparaginase can be L-asparaginase (e.g., L-asparaginase II), which hydrolyzes the amino acid L-asparagine (also known as (S)-2,4-diamino-4-oxobutanoic acid or asparagine or abbreviated as Asn or N) to produce L-aspartic acid (also known as (S)-2-aminosuccinic acid) and ammonia according to the following reaction:

In some cases, asparaginase can hydrolyze the amino acid L-glutamine (also known as (S)-2,5-diamino-5-oxopentanoic acid or abbreviated as Gln or Q) to produce L-glutamate (also known as (S)-2-aminoglutarate) and ammonia according to the following reaction:

The above-mentioned reaction of L-asparaginase mediation can also be referred to as deamination reaction. In some cases, the L-asparaginase in the composition derives from a prokaryotic source, for example, bacteria, including, but not limited to, bacterium Escherichia coli (Escherichia coli (E. coli)). As such, the asparaginase in the subject composition can be an E. coli asparaginase. In some cases, asparaginase is expressed by E. coli. Asparaginase can be recovered and purified from a culture medium comprising the E. coli expressing asparaginase. Except wild-type asparaginase, asparaginase can also be an active fragment of a non-naturally occurring asparaginase and/or a synthetically produced asparaginase and/or a naturally occurring and/or synthetic asparaginase. Examples of asparaginases that can be used in embodiments of the present invention include, but are not limited to, those described in the following documents: 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 by reference.

As mentioned above, the asparaginase in the polyalkylene oxide-asparaginase composition is an asparaginase covalently linked to one or more polyalkylene oxide groups. For example, the asparaginase can include one or more polyalkylene oxide groups, which are covalently linked to the asparaginase by a post-translational modification method. The polyalkylene oxide-asparaginase can include one or more polyalkylene oxide groups, which are covalently linked to the asparaginase at one or more positions on the asparaginase. For example, the polyalkylene oxide group can be covalently linked to an amino acid residue on the asparaginase. In some cases, the polyalkylene oxide group is covalently linked to the amino acid residue of the asparaginase. In some embodiments, the polyalkylene oxide group is covalently linked to the amino acid side chain of the N-terminal amino acid in the asparaginase. In some embodiments, the polyalkylene oxide group is covalently linked to the ε-amino group of lysine (K) in the asparaginase. In some embodiments, the polyalkylene oxide group is covalently linked to the amino acid side chain of the N-terminal amino acid and the ε-amino group of lysine (K) in the asparaginase. In some cases, the polyalkylene oxide-asparaginase is substantially non-antigenic. By "non-antigenic" or "substantially non-antigenic" is meant a composition that does not elicit a significant immune response when the composition is administered to a subject. In some cases, 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, such as 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days or more, or 8 days or more, or 9 days or more, or 10 days or more, or 11 days or more, or 12 days or more, or 13 days or more, or 14 days or more, or 15 days or more, or 16 days or more, or 17 days or more, or 18 days or more, or 19 days or more, or 20 days or more. In some embodiments, the elimination half-life of the polyalkylene oxide-asparaginase can be 3 days or more. In some embodiments, the elimination half-life of the polyalkylene oxide-asparaginase may be 5 days or more.

The polyalkylene oxide group that is connected to asparaginase can be the polyalkylene oxide group of any physiological compatibility.Poly (alkylene oxide) (PAO), also referred to as polyoxyalkylene (POA) is prepared by polymerizing alkylene oxide (for example ethylene oxide, propylene oxide, butylene oxide).Homopolymer is formed by only one type of alkylene oxide, while copolymer is formed by two or more different alkylene oxides, is called alkylene oxide copolymer (AOC).The former example is poly (ethylene oxide) (PEO), it is the polymer of ethylene oxide (EO), and poly (propylene oxide) (PPO) is the polymer of propylene oxide (PO).Poly (ethylene oxide) is also referred to as polyethylene glycol (PEG) or polyoxyethylene (POE) usually.The molecular weight of this type of polymer is usually characterized by the mean value (or repeating unit) of length distribution. In addition to the standard linear form, branched or star-shaped forms of poly(alkylene oxides) are produced by initiating the polymerization reaction with a multifunctional initiator bearing multiple hydroxyl, amino, or sulfhydryl groups, each of which can serve as a starting point for polymer chain growth. For example, the use of glycerol (3 hydroxyl groups) as an initiator produces a 3-arm branched polymer, while pentaerythritol produces a 4-arm polymer. Conventionally, polymers of this type with 3-10 arms are referred to as "branched," while those with more than 10 arms are referred to as "star-shaped" polymers. "Comb-shaped" copolymers are similar to the branched and star-shaped forms, but the initiator for comb-shaped copolymers is a multifunctional polymer bearing multiple hydroxyl, amino, or sulfhydryl groups spaced along the initiator backbone, each of which can serve as a starting point for polymer chain growth. "Grafted" copolymers are prepared by adding polymer side chains or pendant functional groups (e.g., hydroxyl groups) along a polymer backbone with unsaturated C=C bonds, to which the side chains can be added using reactive monofunctional polymer chains. In addition to multiple alkylene oxide-derived repeating units, all poly (alkylene oxides) also contain a single residue equivalent to the initiator polymer synthesis. For linear polymers, it can be an alkylene oxide equivalent to the alkylene oxide used for synthesis (for example, ethylene glycol and ethylene oxide, respectively), and the residue derived from the initiator is no different from the other repeating units on the polymer chain. However, small molecules other than alkylene glycols are commonly used as initiators, examples of which include methanol or N-butanol (for linear polymers) and trimethylolpropane, glycerol and pentaerythritol (for branched polymers) or ethylenediamine. The initiator mass is usually very small compared to the final polymer chain mass and can usually be ignored. Therefore, the term poly (alkylene oxide) is used in this article with its conventional meaning and includes poly (alkylene oxide) initiated with an alkylene glycol molecule and poly (alkylene oxide) initiated with another small molecule.

In certain embodiments, physiologically compatible polyalkylene oxide groups are substantially stable under conditions compatible with living cells, such as primarily aqueous conditions of temperature, pH, salinity, etc., with which they are compatible. In certain embodiments, the polyalkylene oxide groups are water-soluble. The term "water-soluble polymer" refers to a polyalkylene oxide group that is substantially soluble in water, such as aqueous conditions found in a subject's body. Polyalkylene oxide groups of interest include, but are not limited to, linear polyalkylene glycols. The linear polyalkylene glycols used in embodiments of the present invention have the following structural formula:

wherein R is selected from hydrogen, lower alkyl, and mixtures thereof, _R is selected from hydrogen and lower alkyl, and n is a positive integer. By "lower alkyl" is meant an alkyl group having 1 to 4 carbon atoms, i.e., methyl, ethyl, propyl, butyl, and isomers thereof. R may be selected from hydrogen, methyl, and mixtures thereof, _R may be selected from hydrogen and methyl, and n may be a positive integer selected so as to provide the desired polymer size. In some cases, 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 certain embodiments, the polyethylene glycol (PEG) has a molecular weight of 1,000-20,000 Daltons. In some embodiments, the PEG has a molecular weight of 1,000-20,000 Daltons, or 1,000-19,000 Daltons, or 1,000-18,000 Daltons, or 1,000-17,000 Daltons, or 1,000-16,000 Daltons, or 1,000-15,000 Daltons, or 1,000-14,000 Daltons, or 1,000-13,000 Daltons, or 1,000-12,000 Daltons, or 1,000 In certain embodiments, PEG has a molecular weight of 2,000-10,000 daltons. In certain embodiments, PEG has a molecular weight of 4,000-6,000 daltons. In certain embodiments, PEG has a molecular weight of 5,000 daltons. In some cases, the polyethylene glycol is methoxypolyethylene glycol (eg, monomethoxypolyethylene glycol or "mPEG").

As mentioned above, the polyalkylene oxide group can be covalently linked to asparaginase. In some cases, the polyalkylene oxide group is covalently linked to asparaginase via a linking group. As such, the polyalkylene oxide group can be covalently linked to asparaginase via a linking group. The linking group can be any convenient functional group capable of connecting the polyalkylene oxide group to asparaginase. For example, the linking group can include a reactive functional group that provides a covalent bond between the polyoxyethylene group and the asparaginase. In some cases, the linking group can include a reactive functional group that provides a covalent bond between the polyoxyethylene group and the amino acid residue of the asparaginase. For example, the linking group can include a reactive functional group that provides a covalent bond between the polyoxyethylene group and the amino acid residue of the asparaginase. Examples of such reactive functional groups include, but are not limited to, p-nitrophenoxy, thiazolidinylthione, N-hydroxysuccinimide, or other suitable reactive functional groups, such as, but not limited to, N-hydroxybenzotriazole, halogen, N-hydroxyphthalimide, imidazole, O-acylurea, pentafluorophenol, or 2,4,6-trichlorophenol. In some cases, the reactive functional group of the linker is N-hydroxysuccinimide. Thus, after the polyalkylene oxide group is covalently linked to asparaginase, the linker can include functional groups such as, but not limited to, urethane linkers (also known as carbamate linkers), succinate linkers, and the like.

In certain embodiments, the linker comprises a urethane linker (also referred to as a carbamate linker). For example, the reaction of linking methoxypolyethylene glycol (mPEG) to the amino group of an amino acid of a polypeptide (e.g., asparaginase) via a urethane (carbamate) linker is as follows.

In the above reaction, methoxypolyethylene glycol succinimidyl carbonate (also known as SC-PEG) reacts with the amino groups of an amino acid of a polypeptide (e.g., asparaginase) to produce polyethylene glycol-asparaginase with a urethane (also known as carbamate) linker. SC-PEG-asparaginase is also described in Angiolillo, AL. et al., "Pharmacokinetic (PK) and pharmacodynamics (PD) properties of SC-PEG Escherichiacoli 1-asparaginase (EZN-2285) in the treatment of patients with acute lymphoblastic leukemia (ALL): Results from Children's Oncology Group (COG) study AALL07P4", 2012 American Society of Clinical Oncology (ASCO) AnnuaI Meeting, Poster 9543; and Angiolillo, AL. et al., "Pharmacokinetic and Pharmacodynamic Properties of Calaspargase Pegol Escherichia coli L-asparaginase in the Treatment of Patients With Acute Lymphoblastic Leukemia: Results FromChildren's Oncology Group Study AALL07P4", J Clin.Oncology, 32(34), 2014, 3874-3882.

In certain embodiments, the linker comprises a succinate linker (also referred to as a succinyl linker). For example, the reaction of methoxypolyethylene glycol (mPEG) to the amino group of an amino acid of a polypeptide (e.g., asparaginase) via a succinate linker is shown below.

In certain embodiments, methoxypolyethylene glycol succinimidyl succinate (also referred to as SS-PEG) is reacted with the amino group of an amino acid of a polypeptide (e.g., asparaginase) to produce a polyethylene glycol-asparaginase with a succinate linkage. SS-PEG-asparaginase is also described in U.S. Patents 5,122,614; 5,324,844; and 5,612,460, the disclosures of each of which are incorporated herein by reference.

In certain embodiments, the composition comprising polyalkylene oxide-asparaginase is a dehydrated composition. As used herein, a dehydrated composition is a composition that includes a small amount of water, such as 25% or less, or 20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% or less, or 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 water, as measured by Karl Fischer (KF) titration. In some cases, the dehydrated composition has 3% or less water, as measured by Karl Fischer titration. In some cases, the dehydrated composition has 1% or less water, as measured by Karl Fischer titration. In some cases, the dehydrated composition has 0.5% or less water, as measured by Karl Fischer titration. Any convenient protocol can be used to produce the dehydrated composition, such as increasing the temperature of the composition (eg, heating), reducing the pressure, lyophilization (also known as freeze drying), and the like, and combinations thereof.

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 cases, a lyophilized composition is a composition in which water has been removed from the composition by sublimation, wherein the water in the composition undergoes a phase change from a solid to a gas. For example, a lyophilized composition can be a composition in which water has been removed from the composition 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 described above, the lyophilized composition can include a small amount of water, such as 25% or less, or 20% or less, or 15% or less, or 10% or less, or 9% or less, or 8% or less, or 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, as determined by Karl Fischer (KF) titration. In certain embodiments, the lyophilized composition can include a small amount of water, 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 determined by Karl Fischer (KF) titration. In certain embodiments, the lyophilized composition can include a small amount of water, 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 determined by Karl Fischer (KF) titration. In some cases, the lyophilized composition has 3% or less water, as determined by Karl Fischer titration. In some cases, the lyophilized composition has 1% or less water, as determined by Karl Fischer titration. In some cases, the lyophilized composition has 0.5% or less water, as determined by Karl Fischer titration.

Due to the low water content of the lyophilized composition as described above, the lyophilized composition can be in solid form. In some cases, the solid lyophilized composition is a powder. In some cases, the lyophilized composition can be advantageous for storing the composition for an extended period 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), wherein the composition is substantially stable for an extended period of time. By "stable," "storage-stable," or "substantially stable" is meant a composition that does not significantly degrade and/or lose activity over an extended period of time. For example, a storage-stable composition may not have a significant amount of impurities resulting from degradation of the composition over an extended period of time, e.g., 10% or less impurities, or 9% or less, or 8% or less, or 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 degradation products over an extended period of time. In certain embodiments, a storage-stable composition may have less than 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% degradation products over an extended period of time. In some cases, a storage-stable composition may have 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, for example, retains 100% of its activity, 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, for example, 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. For example, the storage-stable composition retains 90% or more of its activity over an extended period of time. In some cases, the storage-stable composition retains 95% or more of its activity over an extended period of time. The extended time limit is a certain time limit, 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, the extended time limit can be 6 months or more. In some cases, the extended time limit is 9 months or more. In some cases, the extended time limit is 1 year (e.g., 12 months) or more. In some cases, the extended time limit is 1.5 years (e.g., 18 months) or more. In some cases, the extended time limit is 2 years (e.g., 24 months) or more. In some embodiments, the extended period of time can be about 1 week to about 3 weeks, or about 1 month to about 6 months, or about 6 months to about 9 months, or about 1 year to about 1.5 years, or about 1 year to about 2 years, or about 1 year to about 3 years, or about 1 year to about 4 years, or about 1 year to about 5 years. In some embodiments, the storage-stable composition is substantially stable for an extended period of time at ambient temperature, for example, a temperature of 20-40° C. or 25-35° C. or 25-30° C. In some cases, the storage-stable composition is substantially stable for an extended period of time at a temperature below ambient temperature, for example, a temperature of 0-20° C. or 0-15° C. or 0-10° C. or 2-8° C.

In some cases, the composition includes a therapeutically effective amount of polyalkylene oxide-asparaginase. The enzymatic activity of polyalkylene oxide-asparaginase can be measured in International Units (IU), which is equivalent to the amount of enzyme required to generate 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 (e.g., the polyalkylene oxide-asparaginase can have a potency (activity)) of 100-5,000 IU/g, such as 500-4,500 IU/g, or 500-4,000 IU/g, or 500-3,500 IU/g, or 500-3,000 IU/g, or 500-2,500 IU/g, or 500-2,000 IU/g, or 500-1,500 IU/g, or 500-1,000 IU/g, or 600-900 IU/g, or 700-800 IU/g. In some cases, the polyalkylene oxide-asparaginase can be present in the composition in an amount of 500-1,000 IU/g. For example, the polyalkylene oxide-asparaginase can have a potency (activity) of 500-1,000 IU/g. In some cases, the polyalkylene oxide-asparaginase is present in the composition in an amount of 700-800 IU/g. For example, the polyalkylene oxide-asparaginase can have a potency (activity) of 700-800 IU/g. In some cases, the polyalkylene oxide-asparaginase can be present in the composition in an amount of 750 IU/g. For example, the polyalkylene oxide-asparaginase can have a potency (activity) of 750 IU/g.

In some cases, the polyalkylene oxide-asparaginase in the composition is present in a therapeutically effective amount, wherein the polyalkylene oxide-asparaginase has a specific activity of 50 IU/mg protein or more, e.g., 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 91 IU/mg protein or more. The specific activity of the polyalkylene oxide-asparaginase in the composition may be 5 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. For example, the polyalkylene oxide-asparaginase in the composition has 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, or 85-115 IU/mg protein, or 90-110 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, e.g., 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 may have a specific activity of 75-125 IU/mg protein.

In certain embodiments, the polyalkylene oxide-asparaginase in the composition is present in a therapeutically effective amount, wherein the polyalkylene oxide-asparaginase in the composition is present in an amount of 1 mg/mL-15 mg/mL, e.g., 1.5 mg/mL-14.5 mg/mL, or 2 mg/mL-14 mg/mL, or 2.5 mg/mL-13.5 mg/mL, or 3 mg/mL-13 mg/mL, or 3.5 mg/mL-12.5 mg/mL, or 4 mg/mL-12 mg/mL, or 4.5 mg/mL-11.5 mg/mL, or 4.5 mg/mL-11 mg/mL, or 4.5 mg/mL-10.5 mg/mL, or 4.5 mg/mL-10 mg/mL, or 4.5 mg/mL-9.5 mg/mL, or 4.5 mg/mL-9 mg/mL or 4.5 mg/mL-8.5 mg/mL, or 5 mg/mL-8 mg/mL. In some cases, the polyalkylene oxide-asparaginase in the composition is present in an amount of 4.5 mg/mL-8.5 mg/mL.

When administered to a subject, the composition can include an amount of polyalkylene oxide-asparaginase sufficient to deliver 100-5,000 IU/m ² of polyalkylene oxide-asparaginase to the subject, e.g., 500-5,000 IU/m ² , or 500-4,500 IU/m ² , or 500-4,000 IU/m 2 , or 500-3,500 IU/m ² , or 500-3,000 IU/m ² , or 1,000-3,000 ^IU /m ² , or 1,500-3,000 IU/m ² , or 1,750-3,000 IU/m ² , or 2,000-3,000 IU/m ² , or 2,000-2,750 IU/m ² , or 2,250-2,750 IU/m ² of polyalkylene oxide-asparaginase. For example, the composition can include a polyalkylene oxide-asparaginase in an amount sufficient to deliver 1,500-3,000 IU/m ² of polyalkylene oxide-asparaginase to a subject. In some cases, the composition includes a polyalkylene oxide-asparaginase in an amount sufficient to deliver 2,000-2,750 IU/m ² of polyalkylene oxide-asparaginase to a subject. In some cases, the composition includes a polyalkylene oxide-asparaginase in an amount sufficient to deliver 2,250-2,750 IU/m ² of polyalkylene oxide-asparaginase to a subject. For example, the composition includes a polyalkylene oxide-asparaginase in an amount sufficient to deliver 2,500 IU/m ² of polyalkylene oxide-asparaginase to a subject.

In certain embodiments, the dose administered to the subject is a liquid dose, eg, an aqueous dose. In some embodiments, the dose includes a buffer and a salt in addition to the polyalkylene oxide-asparaginase.

The compositions of the present disclosure may also include other ingredients in addition to polyalkylene oxide-asparaginase. For example, the compositions may include a buffer. Buffers suitable for compositions of the present disclosure include those compatible with polyalkylene oxide-asparaginase and suitable for, for example, being administered to a subject 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 combinations thereof. The buffer included in the compositions may be a buffer maintaining the pH of the compositions at a physiologically compatible pH, such as a pH of 6-8 or a pH of about 7, such as 7.2, 7.3, or 7.4. In some cases, the buffer is a phosphate buffer. Phosphate buffers may include disodium hydrogen phosphate (also known as disodium phosphate or sodium hydrogen phosphate; Na ₂ HPO ₄ ) and/or sodium dihydrogen phosphate (also known as monosodium phosphate; NaH ₂ PO ₄ ).

In some cases, the amount of sodium phosphate dibasic in the composition is 0.05-5 wt.%, such as 0.1-4.5 wt.%, or 0.1-4 wt.%, or 0.1-3.5 wt.%, or 0.1-3 wt.%, or 0.1-2.5 wt.%, or 0.1-2 wt.%, or 0.1-1 wt.%, or 0.1-0.9 wt.%, or 0.1-0.8 wt.%, or 0.1-0.7 wt.%, or 0.1-0.6 wt.%, or 0.2-0.6 wt.%, or 0.3-0.6 wt.%, or 0.4-0.6 wt.%, or 0.5-0.6 wt.%. For example, sodium phosphate dibasic can be present in the composition in an amount of 0.1-1.0 wt.%. In some cases, sodium phosphate dibasic can be present in the composition in an amount of 0.2-0.8 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.3-0.6 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.5-0.6 wt.%. For example, disodium hydrogen phosphate can 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 sodium dihydrogen phosphate in the composition is 0.005-2 wt.%, such as 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 %. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.05-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.01-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.09-0.15 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.1-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.1-0.15 wt.%. For example, sodium dihydrogen phosphate can be present in the composition in an amount of 0.12 wt.% (or 0.129 wt.%).

Another component that may be present in the compositions of the present disclosure is a salt. Suitable salts for use in the compositions of the present disclosure include salts that are compatible with polyalkylene oxide-asparaginase and 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, and the like, and combinations thereof. In some cases, the salt is sodium chloride.

In some cases, the amount of salt (e.g., sodium chloride) in the composition is 0.05-5 wt.%, e.g., 0.05-4 wt.%, or 0.05-3 wt.%, or 0.05-2 wt.%, or 0.1-5 wt.%, or 0.1-4 wt.%, or 0.1-3 wt.%, or 0.1-2 wt.%, or 0.1-1.5 wt.%, or 0.1-1 wt.%, or 0.2-1 wt.%, or 0.3-1 wt.%, or 0.4-1 wt.%, or 0.5-1 wt.%, or 0.6-1 wt.%, or 0.7-1 wt.%, or 0.8-1 wt.%, or 0.8-0.9 wt.%. For example, salt (e.g., sodium chloride) can be present in the composition in an amount of 0.5-1 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount of 0.2-2 wt.%. In some cases, a salt (e.g., sodium chloride) can be present in the composition in an amount of 0.7-1 wt.%. In some cases, a salt (e.g., sodium chloride) can be present in the composition in an amount of 0.8-0.9 wt.%. For example, a salt (e.g., sodium chloride) can be present in the composition in an amount of 0.85 wt.%.

In certain embodiments, the composition comprising polyalkylene oxide-asparaginase is a lyophilized composition. The lyophilized compositions of the present disclosure may further comprise a buffer, a salt, and a sugar in addition to the polyalkylene oxide-asparaginase. For example, aspects of the present disclosure include a lyophilized, storage-stable composition of polyalkylene oxide-asparaginase comprising a polyalkylene oxide covalently linked to the asparaginase via a linker, a buffer, a salt, and a sugar.

Buffers suitable for freeze-dried compositions of the present disclosure include those compatible with polyalkylene oxide-asparaginase and suitable for, for example, administration to a subject by injection or intravenous administration. Examples of suitable buffers include those as described above. In some cases, the buffer is a phosphate buffer. In certain embodiments, the phosphate buffer includes sodium hydrogen phosphate and sodium dihydrogen phosphate. In some cases, the amount of sodium hydrogen phosphate in the composition is 0.05-1wt.%, such as 0.1-0.9wt.%, or 0.1-0.8wt.%, or 0.1-0.7wt.%, or 0.1-0.6wt.%, or 0.1-0.5wt.%, or 0.1-0.4wt.%, or 0.2-0.4wt.%, or 0.2-0.3wt.%, or 0.25-0.3wt.%. For example, sodium hydrogen phosphate can be present in the composition in an amount of 0.1-0.5wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.2-0.4 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.25-0.3 wt.%. For example, disodium hydrogen 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 sodium dihydrogen phosphate in the composition is 0.005-1 wt.%, e.g., 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 0.01-0.2 wt.%, or 0.01-0.1 wt.%, 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, sodium dihydrogen phosphate can be present in the composition in an amount of 0.01-0.1 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.05-0.07 wt.%. In some cases, sodium dihydrogen phosphate can be present in the composition in an amount of 0.045-0.075 wt.%. For example, sodium dihydrogen phosphate can be present in the composition in an amount of 0.06 wt.%.

In some aspects, 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 suitable for administration to a subject, for example, by injection or intravenous administration. Examples of suitable salts include those described above. In some cases, the salt is sodium chloride.

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

In some cases, the amount of sugar (such as sucrose) in the composition is 0.1-25wt.%, such as 0.5-20wt.%, or 1-15wt.%, or 1-10wt.%, or 1-9wt.%, or 1-8wt.%, or 2-7wt.%, or 2-6wt.%, or 3-5wt.%, or 4-5wt.%. For example, sugar (such as sucrose) can be present in the composition in an amount of 1-10wt.%. In some cases, sugar (such as sucrose) can be present in the composition in an amount of 3-5wt.%. In some cases, sugar (such as sucrose) can be present in the composition in an amount of 4-5wt.%. For example, sugar (such as sucrose) can be present in the composition in an amount of 4.5wt.%.

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

In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 700-800 IU/g, disodium hydrogen phosphate in an amount of 0.2-0.8 wt.%, sodium dihydrogen phosphate in an amount of 0.1-0.14 wt.%, a salt (e.g., sodium chloride) in an amount of 0.6-1.0 wt.%, and water.

In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 700-800 IU/g, disodium hydrogen phosphate in an amount of 0.5-0.6 wt.%, sodium dihydrogen phosphate in an amount of 0.09-0.15 wt.%, a salt (e.g., sodium chloride) in an amount of 0.8-0.9 wt.%, and water.

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

In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 750 IU/g, disodium hydrogen phosphate in an amount of 0.56 wt.% (or 0.558 wt.%), sodium dihydrogen phosphate in an amount of 0.13 wt.% (or 0.129 wt.%), a salt (e.g., sodium chloride) in an amount of 0.85 wt.%, and water.

In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase, disodium hydrogen phosphate, sodium dihydrogen phosphate, a salt (e.g., sodium chloride), and water. In other embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase, disodium hydrogen phosphate, sodium dihydrogen phosphate, a salt (e.g., sodium chloride), a sugar (e.g., sucrose), and water.

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

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

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

In some embodiments, the composition comprises, consists essentially of, or consists of polyalkylene oxide-asparaginase having a potency (activity) of 750 IU/g, disodium hydrogen phosphate in an amount of 0.3 wt.%, sodium dihydrogen phosphate in an amount of 0.06 wt.%, a salt (e.g., sodium chloride) in an amount of 0.4 wt.%, a sugar (e.g., sucrose) in an amount of about 4.5 wt.%, and water.

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

In some cases, the composition (e.g., liquid or lyophilized composition) is a sterile composition. "Sterile" refers to a composition that is substantially free of immunogenic components, such as substantially free of microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.). In some cases, the composition is present in a container. Providing a composition in a container can help the composition become a sterile composition. For example, the container can be configured to maintain the composition encapsulated in a sterile environment in the container. In this manner, the container can be a sealed container, such as a container that can include a seal, such as a waterproof and/or airtight seal. The seal can be removed from the container to allow the user to access the container contents. In some cases, the seal can be a brittle seal, or in other cases, the seal can be configured to allow a needle, cannula, or syringe to be inserted into the container interior without removing the seal from the container. In some cases, the seal configured allows entry into the container interior, but without removing the seal from the container, it can be beneficial to maintain the container contents (e.g., the composition in the container) in a sterile environment before the composition is administered to the subject. In some embodiments, sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like. The sealant can be used to seal a sample or a mixture of the sample and the like.

In some cases, the container is a unit dose container. A unit dose container refers to a container containing one or more unit doses to be administered to a subject. In some embodiments, a unit dose container includes a predetermined amount of the subject composition calculated to be sufficient to produce the desired effect of 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 the active composition administered to the subject depends on the subject to be treated, the severity of the patient, and the mode of administration. For example, a unit dose container can include the amount of the composition administered as an effective amount to achieve the desired effect in the subject to be treated as disclosed herein. In some cases, a unit dose container includes a composition with a therapeutically effective amount of polyalkylene oxide-asparagine. The therapeutically effective amount of polyalkylene oxide-asparagine is as 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 in relation to the sealed container).

The container can be made of any convenient material compatible with the polyalkylene oxide-asparaginase and the 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 cases, the container is a liquid-compatible container configured to contain a liquid. The container can also be solid- and liquid-compatible, wherein the container is configured to contain a solid and a liquid. In some cases, the liquid in the container can be an aqueous liquid, and in these cases, the container can be compatible with the aqueous composition. The so-called "compatibility" means that the container is substantially inert (e.g., does not significantly react with) the liquid and/or the composition or other components that contact the container. Examples of suitable container materials include, but are not limited to, glass and plastic. For example, the container can be made of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX ^™ ), fused silica glass, fused silica glass, etc. Other examples of suitable container materials for the container include plastics such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethyl ether (PFE), Teflon (FEP), perfluoroalkoxyalkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), polystyrene, etc. In some cases, as described above, the container is a vial, and as such, can be a glass vial. As described above, the container can be a sealed container, and as such, can be a sealed glass vial.

As described in more detail below, the liquid of the present disclosure or the composition of reconstruction can be administered to a subject by injection or intravenously. In certain embodiments, before administering the reconstructed composition to the subject, for example, as described above, the solid composition can be merged with a liquid to obtain a liquid composition suitable for, for example, by injection or intravenous administration. In some cases, before the composition is administered to the subject, the solid composition can be merged with water (for example, water for injection, WFI) to obtain an aqueous composition suitable for, for example, by injection or intravenous administration. For example, a lyophilized composition can be reconstructed with water (for example, water for injection, WFI) to produce a dosage unit suitable for, for example, by injection or intravenous administration.

As described above, aspects of the present disclosure include compositions comprising: a polyalkylene oxide-asparaginase comprising a polyalkylene oxide covalently linked to an asparaginase via a linker; a buffer and a salt. In certain embodiments, as described above, the polyalkylene oxide is polyethylene glycol. In certain embodiments, as described above, the linker is a urethane (carbamate) linker. In certain embodiments, as described above, the asparaginase is an E. coli asparaginase. In certain embodiments, as described above, the buffer is a phosphate buffer. In certain embodiments, as described above, the salt is sodium chloride. Therefore, certain embodiments of the composition include polyethylene glycol-asparaginase comprising a polyethylene glycol group linked to an E. coli asparaginase via urethane; a phosphate buffer and a salt. Each of these composition components (e.g., the molecular weight of polyethylene glycol, the amount of polyethylene glycol-asparaginase, the amount and type of phosphate buffer, the amount and type of salt) is as described in detail above.

As described above, aspects of the present disclosure include freeze-dried storage-stable compositions comprising: an alkylene oxide-asparaginase comprising a polyalkylene oxide group covalently linked to an asparaginase via a linker; a buffer, a salt, and a sugar. In certain embodiments, as described above, the polyalkylene oxide is polyethylene glycol. In certain embodiments, as described above, the linker is a urethane (carbamate) linker. In certain embodiments, as described above, the asparaginase is an E. coli asparaginase. In certain embodiments, as described above, the buffer is a phosphate buffer. In certain embodiments, as described above, the salt is sodium chloride. In certain embodiments, as described above, the sugar is a disaccharide (e.g., sucrose). Therefore, certain embodiments of the freeze-dried storage-stable composition include polyethylene glycol-asparaginase comprising a polyethylene glycol group linked to an E. coli asparaginase via an urethane; a phosphate buffer, a salt, and a disaccharide. Each of these composition components (eg, the molecular weight of polyethylene glycol, the amount of polyethylene glycol-asparaginase and the type of phosphate buffer, the amount and type of salt, the amount and type of disaccharide) is described in detail above.

Compositions of the present disclosure may also include other ingredients, such as other pharmaceutically acceptable excipients or dosage delivery vehicles as components of the composition. Excipients may include, but are not limited to, carbohydrates, inorganic salts, organic salts, antimicrobials, antioxidants, surfactants. Water (e.g., water for injection (WFI)), alcohol, polyols, glycerol, vegetable oils, phospholipids, buffers, alkalis, and combinations thereof. Carbohydrates, such as sugars, derived sugars, such as alditols, uronic acids, esterified sugars, and/or sugar polymers may also be used. Some carbohydrate excipients of interest include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, inositol, etc. Inorganic and organic salts may include, but are not limited to, citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, and any combination thereof.

In certain embodiments, the compositions of the present disclosure may further include an antimicrobial agent for preventing or inhibiting the growth of microorganisms, such as benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and any combination thereof.

One or more antioxidants may also be included in the composition. Antioxidants that can reduce or prevent oxidation and thus the deterioration of the composition may include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, dihydroxypropyl mercaptan, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and any combination thereof.

One or more surfactants can also be included in the compositions of the present disclosure. For example, suitable surfactants include, but are not limited to, polysorbates, such as "Tween 20" and "Tween 80" and poloxamers, such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids, such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, fatty acids and fatty acid esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other cations.

Acids or bases may also be present in the compositions of the present disclosure. For example, acids may 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, phosphoric 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.

The amount of any single excipient in the composition can vary according to the specific needs of the properties and functions of the excipient, the dosage delivery vehicle and the composition. In some cases, the optimum amount of any single excipient is determined by routine experimentation, i.e., by preparing compositions containing different amounts of excipients (ranging from low to high), checking stability and other parameters, and then determining the range therein, which can achieve optimal performance without obvious adverse effects. However, typically, the amount of excipient in the composition is 1%-99% by weight of the excipient, for example 5%-98% by weight, for example 15%-95% by weight, including 30% or less by weight, or 20% or less by weight, or 10% or less by weight. Pharmaceutical excipients, as well as other excipients that can be used in the compositions, are described in “Remington: The Science & Practice of Pharmacy,” 22nd ed., Williams & Williams, (2012), “Physician's Desk Reference,” 70th ed., PDR Network, Montvale, NJ (2015), and Rowe, RC., Handbook of Pharmaceutical Excipients, 7th ed., Pharmaceutical Press, New York, NY, (2012), the disclosures of each of which are incorporated herein by reference.

How to use

Aspects of the present disclosure also include methods of using the compositions described herein (e.g., liquid and lyophilized). In certain embodiments, the method of use is a method of deaminating asparagine in a subject. As described above, asparaginase can mediate a deamination reaction in which the amino acid asparagine is hydrolyzed to generate aspartic acid and ammonia, for example, according to the following reaction:

In some cases, the activity of asparaginase reduces the concentration of asparagine in the subject, such as reducing the plasma concentration of asparagine in the subject. The consumption of asparagine in the subject may have an adverse effect on the cells that rely on the presence of asparagine for protein synthesis in the subject. For example, protein synthesis in cells that lack the ability to synthesize their own asparagine (such as cells lacking asparagine synthetase) may be adversely affected by the lack of exogenous asparagine, and the latter may cause apoptosis. In some cases, cells that rely on the presence of asparagine to perform protein synthesis in the subject may be associated with tumor conditions, such as cancer. Therefore, the method of the present disclosure includes a method for treating a tumor condition in the subject, such as a method for treating a cancer in the subject. Therefore, the present disclosure includes a composition that polyalkylene oxide-asparaginase may be effective for treating tumor conditions, such as cancer. In certain embodiments, the non-tumor cells in the subject will not be significantly affected by the polyalkylene oxide-asparagine of the present disclosure. For example, the non-tumor cells in the subject can have the enzyme asparagine synthetase, and thus maintain the ability to synthesize asparagine.

In certain embodiments, the neoplastic condition to be treated in the subject includes a neoplastic condition amenable to treatment by administering polyalkylene oxide-asparaginase to the subject, such as a neoplastic condition that is dependent on exogenous asparagine. For example, a neoplastic condition treatable by administering polyalkylene oxide-asparaginase to the subject includes cancer, such as a solid tumor or a liquid tumor.

In some cases, the neoplastic condition is characterized by the presence of a solid tumor. Therefore, in some embodiments, the method of the present disclosure is a method of treating a subject with a solid tumor using a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure). The method of treating a subject's neoplastic condition can be used to treat a variety of solid tumors, including carcinomas and sarcomas. The types of solid tumors may include, but are not limited to, pancreatic cancer, melanoma, squamous cell carcinoma, non-squamous cell lung cancer (NSCLC), colon cancer, breast cancer, ovarian cancer, cervical cancer, prostate cancer, and the like. For example, cancers that can be treated using the subject methods include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (of various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchogenic carcinoma, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell carcinoma and non-small cell carcinoma of the lung, 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 cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteoblastic cancer, epithelial cancer, and nasopharyngeal cancer, among others.

Sarcomas that may be treated using the subject methods include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using the subject methods include, but are not limited to, gliomas, astrocytomas, medulloblastomas, craniopharyngiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, melanomas, neuroblastomas, and retinoblastomas.

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

Various types of leukemia can be treated using the methods of the present invention. For example, the leukemia can be acute leukemia. Acute leukemia can be characterized by a rapid increase in the number of immature blood cells. The rapid increase in immature blood cells can lead to crowding, which can cause the bone marrow to produce significantly less healthy blood cells. Therefore, the methods of the present disclosure include methods for treating a subject with acute leukemia, for example, 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 the subject's acute leukemia.

In other cases, the leukemia is a chronic leukemia. In some cases, chronic leukemia is characterized by an increase in the number of relatively mature but abnormal white blood cells. Chronic leukemia may take a long time to develop (e.g., months or years), wherein the abnormal white blood cells are produced at a significantly higher rate than normal. Therefore, the methods of the present disclosure include methods for treating chronic leukemia in a subject, for example, by administering to the subject a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) in an amount effective to treat the chronic leukemia in the subject.

In certain embodiments, leukemia is lymphocytic leukemia (also referred to as lymphocytic leukemia). Lymphocytic leukemia may be characterized by the fact that blood type cells are affected by leukemia. In lymphocytic leukemia, abnormal changes in blood cells occur in bone marrow cells that normally develop into lymphocytes. For example, lymphocytic leukemia may be B cell leukemia. Therefore, the method of the present disclosure includes a method for treating a subject with lymphocytic leukemia (lymphocytic leukemia), for example, by administering polyalkylene oxide-asparaginase (e.g., liquid or reconstructed freeze-dried polyalkylene oxide-asparaginase compositions of the present invention) to the subject to effectively treat the subject with lymphocytic leukemia (lymphocytic leukemia). For example, specific types of lymphoblastic leukemia that can be treated using the methods of the present invention include acute lymphoblastic leukemia (ALL). In these embodiments, the methods of the present disclosure include methods for treating a subject with acute lymphoblastic leukemia (ALL), for example, by administering to the subject a dose of a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present invention) effective to treat the subject with ALL. Other types of lymphocytic leukemias treated using the subject methods include, but are not limited to, chronic lymphocytic leukemia (CLL). In these embodiments, the methods of the present disclosure include methods for treating a subject with chronic lymphocytic leukemia (CLL), for example, by administering to the subject a dose of a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) effective to treat the subject with CLL.

In other embodiments, the leukemia is myeloid leukemia (also referred to as myeloid leukemia). Myeloid leukemia is characterized by the type of 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. Therefore, the method of the present disclosure includes a method for treating a subject with myeloid leukemia (myeloid leukemia), for example, by administering to the subject a polyalkylene oxide-asparaginase composition (e.g., a liquid or reconstructed freeze-dried polyalkylene oxide-asparaginase) of a dosage effective to treat the subject's myeloid leukemia (myeloid leukemia). For example, a specific type of myeloid leukemia, which can be treated using the subject method, includes acute myeloid leukemia (AML). In these embodiments, the method of the present disclosure includes a method for treating a subject with acute myeloid leukemia (AML), for example, by administering to the subject a polyalkylene oxide-asparaginase (e.g., a liquid or reconstructed freeze-dried polyalkylene oxide-asparaginase composition) of a dosage effective to treat the subject's AML.

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 AML with t(8;21)(q22;q22), AML1(CBF-α)/ETO, acute promyelocytic leukemia (AML with t(15;17)(q22;qll-12) and variants, PML/RAR-α), AML with abnormal bone marrow eosinophils (inv(16)(p13q22) or t(16;16)(p13;ql1), CBFb/MYH11X, and AML with lqq23 abnormalities (MLL). Examples of AML with multilineage dysplasia may include those associated with or unrelated to a previous myelodysplastic syndrome. Other types of acute myeloid leukemia include, for example, AML minimally differentiated, AML amature, AML mature, acute monocytic leukemia, acute monocytic leukemia, acute erythroid leukemia, acute megakaryocytic leukemia, acute basophilic leukemia, and acute panmyelosis with myelofibrosis.

Other types of myeloid leukemias that can be treated using the subject methods include, but are not limited to, chronic myeloid leukemia (CML). In these embodiments, the methods of the present disclosure include methods for treating chronic myeloid leukemia (CML) in a subject, for example, by administering to the subject a dose of a polyalkylene oxide-asparaginase (e.g., a liquid or reconstituted lyophilized polyalkylene oxide-asparaginase composition of the present disclosure) effective to treat the subject's CML.

In certain instances, asparaginase can mediate the deamination of glutamine, wherein the amino acid glutamine is hydrolyzed to glutamate and ammonia, for example according to the following reaction:

In some cases, the activity of asparaginase reduces the concentration of glutamine in the subject, such as reducing the plasma concentration of glutamine in the subject. Similar to the above, glutamine depletion in the subject can adversely affect cells in the subject that rely on the presence of glutamine for protein synthesis. For example. Protein synthesis in cells that lack the ability to synthesize their own glutamine (e.g., cells lacking the enzyme glutamine synthetase) can be adversely affected by the lack of exogenous glutamine, which can lead to cell apoptosis. In some cases, cells in the subject that rely on the presence of glutamine for protein synthesis may be associated with tumor conditions, such as cancer. Therefore, the methods of the present disclosure include methods for treating tumor conditions in the subject, such as methods for treating cancer in the subject, wherein the tumor condition can rely on exogenous glutamine. For example, embodiments of the subject methods include methods for deaminating glutamine in the subject. In certain embodiments, non-tumor cells in the subject are not significantly affected by the polyalkylene oxide-asparaginase administered to the subject. For example, non-tumor cells in the subject can have glutamine synthetase and thus retain the ability to synthesize glutamine.

As mentioned above, compositions of the present disclosure include freeze-dried storage-stable compositions. Before applying the compositions to the subject, the freeze-dried compositions can be merged with liquid to obtain a liquid composition suitable for, for example, passing through an injection or intravenous administration. In some cases, before applying the compositions to the subject, the freeze-dried compositions can be merged with water (e.g., water for injection, WFI) to obtain an aqueous composition suitable for, for example, passing through an injection or intravenous administration. For example, the method of the present disclosure can include reconstructing the freeze-dried compositions of the present disclosure (e.g., freeze-dried storage-stable compositions). Reconstructing the freeze-dried compositions can produce reconstructed dosage units. In some cases, the reconstructed dosage units are suitable for application to the subject, for example, by injection or intravenous administration. In certain embodiments, reconstructing the freeze-dried compositions includes merging freeze-dried compositions (e.g., freeze-dried storage-stable compositions) and water (e.g., water for injection, WFI).

Liquid or reconstituted dosage units can include a predetermined amount of the present invention's composition calculated to effectively produce a desired therapeutic effect in a subject. The amount of the composition (e.g., liquid or reconstituted) administered to a subject in a dosage unit can depend on the subject to be treated, the severity of the patient, and the mode of administration. For example, a dosage unit can include an amount administered as a composition disclosed herein in a therapeutically effective amount.

Certain embodiments of the dosage unit can include an amount of polyalkylene oxide-asparaginase that is 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 500-2,500 IU/mL, or 500-2,000 IU/mL, or 500-1,500 IU/mL, or 500-1,000 IU/mL, or 600-900 IU/mL, or 700-800 IU/mL. In some cases, the dosage unit includes an amount of polyalkylene oxide-asparaginase that is 500-1,000 IU/mL. In some cases, the dosage unit includes an amount of polyalkylene oxide-asparaginase that is 700-800 IU/mL. For example, a dosage unit can include 750 IU/mL of polyalkylene oxide-asparaginase.

In certain embodiments, a dosage unit comprises a therapeutically effective amount (e.g., specific activity) of polyalkylene oxide-asparaginase, e.g., 50 IU/mg protein or more, e.g., 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. [00145] In some embodiments, the dosage unit may have a specific activity of 85 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. For example, a dosage unit may have a specific activity of 85 IU/mg protein or more. In some embodiments, the dosage unit 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, 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 of 50-150 IU/mg protein, for example, 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 dosage unit can have a specific activity of 75-125 IU/mg protein.

In certain embodiments, a dosage unit comprises a therapeutically effective amount (e.g., protein concentration) of polyalkylene oxide-asparaginase in an amount of 1 mg/mL to 15 mg/mL, e.g., 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, or 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. In some cases, a dosage unit includes polyalkylene oxide-asparaginase in an amount of 4.5 mg/mL-8.5 mg/mL.

When administered to a subject, a dosage unit can include a therapeutically effective amount of polyalkylene oxide-asparaginase such that the dosage unit delivers 100-5,000 IU/m ² of polyalkylene oxide-asparaginase to the subject, e.g., 500-5,000 IU/m ² , or 500-4,500 IU/m 2 , or 500-4,000 IU/m ² , or 500-3,500 IU/m ² , or 500-3,000 ^IU /m ² , or 1,000-3,000 IU/m ² , or 1,500-3,000 IU/m ² , or 1,750-3,000 IU/m ² , or 2,000-3,000 IU/m ² , or 2,000-2,750 IU/m ² , or 2,250-2,750 IU/m ² of polyalkylene oxide-asparaginase. For example, the dosage unit can deliver 1,500-3,000 IU/m ² of polyalkylene oxide-asparaginase to the subject. In some cases, the dosage unit delivers 2,000-2,750 IU/m ² of polyalkylene oxide-asparaginase to the subject. In some cases, the dosage unit delivers 2,250-2,750 IU/m ² of polyalkylene oxide-asparaginase to the subject. For example, the dosage unit can deliver 2,500 IU/m ² of polyalkylene oxide-asparaginase to the subject.

In certain embodiments, the dosage unit includes a buffer, such as the buffers described in detail above. For example, the dosage unit can include a phosphate buffer, and as such can include disodium hydrogen phosphate and sodium dihydrogen phosphate.

In some cases, the dosage unit includes a phosphate buffer and can include sodium phosphate dibasic and sodium dihydrogen phosphate as such. In some cases, the dosage unit includes sodium phosphate dibasic in an amount of 0.5-10 mg/g, for example, 1-9 mg/g or 1-8 mg/g, or 1-7 mg/g, or 2-7 mg/g, or 3-6 mg/g, or 4-6 mg/g, or 5-6 mg/g. For example, the dosage unit can include sodium phosphate dibasic in an amount of 4-6 mg/g. In some cases, the dosage unit can include sodium phosphate dibasic in an amount of 5-6 mg/g. For example, the dosage unit can include sodium phosphate dibasic in an amount of about 5.5 mg/g, for example, 5.6 mg/g (or 5.58 mg/g). In certain embodiments, the dosage unit can include the sodium dihydrogen phosphate of the amount of 0.05-5mg/g, for example, 0.1-4.5mg/g, or 0.1-4mg/g, or 0.1-3.5mg/g, or 0.1-3mg/g, or 0.1-2.5mg/g, or 0.1-2mg/g, or 0.5-2mg/g, or 1-2mg/g, or 1-1.5mg/g. For example, the dosage unit can include the sodium dihydrogen phosphate of the amount of 1-2mg/g. In some cases, the dosage unit can include the sodium dihydrogen phosphate of the amount of 1-1.5mg/g. For example, the dosage unit can include the sodium dihydrogen phosphate of the amount of 1.2mg/g (or 1.29mg/g).

In certain embodiments, the dosage unit includes a salt. Suitable salts for the dosage unit include salts that are compatible with polyalkylene oxide-asparaginase and suitable for, for example, administration to a subject by injection or intravenous administration. 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 some cases, dosage unit includes salt, for example sodium chloride.In some cases, dosage unit includes salt (for example sodium chloride), and its amount is 1-20mg/g, for example 1-19mg/g or 1-18mg/g or 1-17mg/g or 1-16mg/g or 1-15mg/g or 2-15mg/g or 3-15mg/g or 4-15mg/g or 5-14mg/g or 5-13mg/g or 5-12mg/g or 5-11mg/g or 5-10mg/g or 5-9mg/g or 6-9mg/g or 7-9mg/g or 8-9mg/g.For example, dosage unit can include salt (for example sodium chloride), and its amount is 1-10mg/g.In some cases, dosage unit includes salt (for example sodium chloride), and its amount is 5-10mg/g. In some cases, the dosage unit includes a salt (e.g., sodium chloride) in an amount of 6-9 mg/g. In some cases, the dosage unit includes a salt (e.g., sodium chloride) in an amount of 8-9 mg/g. For example, a dosage unit can include a salt (e.g., sodium chloride) in an amount of 8.5 mg/g.

In some cases, the formulation administered to a subject is PEG-asparaginase liquid injection, known under the trade name PEG-asparaginase, which has been approved for commercial sale by the U.S. Food and Drug Administration. (Pegaspargase) is an L-asparaginase (L-asparagine amidohydrolase) covalently conjugated to monomethoxypolyethylene glycol (mPEG) and is presented as a clear, colorless, preservative-free, isotonic sterile solution in phosphate-buffered saline, pH 7.3. Each milliliter contains 750 ± 150 international units of pegaspargase, 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.

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

In certain embodiments, the dosage unit of reconstruction includes a buffer, such as the buffer described in detail above. In some cases, the dosage unit of reconstruction includes sodium hydrogen phosphate in an amount of 0.5-10 mg/g, such as 1-9 mg/g, or 1-8 mg/g, or 1-7 mg/g, or 1-6 mg/g, or 1-5 mg/g, or 1-4 mg/g, or 2-4 mg/g, or 2-3 mg/g, or 2.5-3 mg/g. For example, the dosage unit of reconstruction may include sodium hydrogen phosphate in an amount of 1-5 mg/g. In some cases, the dosage unit of reconstruction may include sodium hydrogen phosphate in an amount of 2-4 mg/g. In some cases, the dosage unit of reconstruction may include sodium hydrogen phosphate in an amount of 2.5-3 mg/g. For example, the dosage unit of reconstruction may include sodium hydrogen phosphate in an amount of about 3 mg/g, such as 2.8 mg/g (or 2.79 mg/g). In certain embodiments, the reconstructed dosage unit can include sodium dihydrogen phosphate in an amount of 0.05-1 mg/g, such as 0.1-0.9 mg/g, or 0.1-0.8 mg/g, or 0.2-0.8 mg/g, or 0.3-0.8 mg/g, or 0.4-0.8 mg/g, or 0.45-0.75 mg/g, or 0.5-0.7 mg/g. For example, the reconstructed dosage unit can include sodium dihydrogen phosphate in an amount of 0.45-0.75 mg/g. In some cases, the reconstructed dosage unit can include sodium dihydrogen phosphate in an amount of 0.5-0.7 mg/g. For example, the reconstructed dosage unit can include sodium dihydrogen phosphate in an amount of 0.6 mg/g.

In certain embodiments, the dosage unit of reconstruction includes salt, such as the salt described in detail above. In some cases, the dosage unit of reconstruction includes salt, such as sodium chloride. In some cases, the dosage unit of reconstruction includes salt (such as sodium chloride) in an amount of 0.5-10mg/g, such as 1-9mg/g or 1-8mg/g or 1-7mg/g or 1-6mg/g or 1-5mg/g or 2-5mg/g or 3-5mg/g or 4-5mg/g or 4-4.5mg/g. For example, the dosage unit of reconstruction can include salt (such as sodium chloride) in an amount of 1-10mg/g. In some cases, the dosage unit of reconstruction can include salt (such as sodium chloride) in an amount of 3-5mg/g. In some cases, the dosage unit of reconstruction includes salt (such as sodium chloride) in an amount of 4-4.55mg/g. For example, the dosage unit of reconstruction includes salt (such as sodium chloride) in an amount of about 4mg/g, such as 4.25mg/g.

In certain embodiments, the dosage unit of reconstruction includes sugar, such as the sugar described in detail above.In some cases, the dosage unit of reconstruction includes sugar, such as disaccharides.In some cases, the dosage unit of reconstruction includes sugar, such as sucrose.In some cases, the dosage unit of reconstruction includes sugar (such as sucrose) of the amount of 1-250mg/g, such as 5-200mg/g or 10-150mg/g or 10-100mg/g or 10-90mg/g or 10-80mg/g or 20-70mg/g or 20-60mg/g or 30-50mg/g or 40-50mg/g.For example, the dosage unit of reconstruction can include sugar (such as sucrose) of the amount of 10-100mg/g.In some cases, the dosage unit of reconstruction can include sugar (such as sucrose) in the amount of 30-50mg/g.In some cases, the dosage unit of reconstruction can include sugar (such as sucrose) in the amount of 40-50mg/g. For example, a reconstituted dosage unit can include sugar (eg, sucrose) in an amount of 45 mg/g.

In certain embodiments, the dosage unit (e.g., liquid or reconstituted) has a pH compatible with physiological conditions. In some cases, the pH of the dosage unit is 6-8. In some cases, the pH of the dosage unit is 7-8. For example, the pH of the dosage unit is 7-7.5. 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.

In certain embodiments, the above methods may include administering a dosage unit to a subject to deaminize asparagine in the subject. The route of administration may be selected based on various factors, including, but not limited to, the condition to be treated, the type of composition and/or the device used, the patient to be treated, etc. The routes of administration for the disclosed methods include, but are not limited to, oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), intramuscular (im), rectal, topical, ocular, nasal, and transdermal. For example, a composition suitable for injection can be administered intravenously, intramuscularly, intradermally, subcutaneously, sublingually, intraosseously, or by other routes of administration.

In some cases, administering the dosage unit to the subject comprises administering the dosage unit to the subject intravenously. In some cases, administering the dosage unit to the subject comprises administering the dosage unit to the subject intramuscularly.

In some cases, administering the reconstituted dosage unit to the subject comprises administering the reconstituted dosage unit to the subject intravenously. In some cases, administering the reconstituted dosage unit to the subject comprises administering the reconstituted dosage unit to the subject intramuscularly.

In certain embodiments, the method includes administering the dosage unit to the subject according to a treatment regimen. For example, in some cases, the subject to be treated can be prescribed by a healthcare provider. In some cases, the treatment regimen includes, but is not necessarily limited to, 5 times a day, 4 times a day, 3 times a day, 2 times a day, 1 time a day, 3 times a week, 2 times a week, 1 time a week, 1 time every 2 weeks, 1 time every 3 weeks, 1 time every month, 1 time every 5 weeks, 1 time every 6 weeks, 1 time every 7 weeks, 1 time every other month, and combinations thereof.

In some embodiments, the treatment regimen includes administering one or more dosages within the extended time limit. In some cases, a single dose (such as a single dose unit) is administered to the subject, and after the initiating dose, one or more dosages are administered to the subject at a subsequent time. In some cases, more than one dosage (such as one or more dosage units) is administered to the subject, and after the initiating dose, one or more dosages are administered to the subject at a subsequent time. For example, a single dose (such as a single dose unit) can be administered to the subject, and after the single dose, a single dose is administered to the subject at a subsequent time point. Other single doses can be administered in time at a subsequent time. In other cases, a single dose (such as a single dose unit) can be administered to the subject, and after the single dose, two dosages can be administered to the subject at a subsequent time. Other single doses or multiple doses can be administered in time at a subsequent time point.

In some cases, the treatment regimen includes multiple phases. The treatment regimen may include multiple phases, wherein the dosage regimen is different in each phase of the treatment regimen. In some cases, the subject is prescribed a treatment regimen with two phases: an induction phase and a consolidation phase. In some cases, the subject is prescribed a treatment regimen with two phases, wherein the dosage regimen of the first phase is different from the dosage regimen in the second phase. For example, the subject may be prescribed a treatment regimen with two phases, including an induction phase and a consolidation phase, wherein the dosage regimen of the induction phase is different from the dosage regimen of the consolidation phase. In another embodiment, the subject may be prescribed a treatment regimen with three phases: an induction phase, a consolidation phase, and a maintenance phase. In some cases, the subject may be prescribed a treatment regimen with three phases, including an induction phase, a consolidation phase, and a maintenance phase, wherein the dosage regimen of each phase is different from the dosage regimen of the other phase. For example, the subject may be prescribed a treatment regimen with three phases, including an induction phase, a consolidation phase, and a maintenance phase, wherein the dosage regimens of the induction phase, the consolidation phase, and the maintenance phase are each different from each other.

In certain embodiments, the treatment duration of each period of the treatment regimen is the same, or in other cases can be different. For example, the time period of the induction period can be 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. In some cases, the time period of the induction period is 4 weeks. In some cases, the time period of the induction period is 5 weeks.

In certain embodiments, the time period during the consolidation period is 1 week or more, e.g., 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 cases, the time period during the consolidation period is 8 weeks. In some cases, the consolidation period is 27 weeks. In some cases, the consolidation period is 30 weeks.

In certain embodiments, the time period of the maintenance period is 1 week or more, e.g., 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 In some cases, the maintenance period is 8 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. In some cases, the maintenance period is 8 weeks. In some cases, the maintenance period is 88 weeks. In some cases, the maintenance period is 104 weeks. In some cases, the maintenance period is 140 weeks. In some cases, the maintenance period is 156 weeks. In some cases, the maintenance period is 88-104 weeks. In some cases, the maintenance period is 88-140 weeks. In some cases, the maintenance period is 88-156 weeks.

The example of the treatment regimen that can be applied to subject includes but is not limited to treatment regimen as described herein.In certain embodiments, treatment regimen includes applying single dosage unit to subject during induction phase and applying multiple dosage units during maintenance phase.In certain embodiments, treatment regimen includes applying single dosage unit to subject during induction phase and applying multiple dosage units to subject during consolidation phase.For example, multiple dosage units can be applied to subject (for example during consolidation phase) by applying dosage unit to subject every 3 weeks.In some cases, single dosage unit is applied to subject every 3 weeks.As mentioned above, consolidation phase can be 30 weeks, therefore 10 dosage units in total (for example, single dosage unit can be applied once every 3 weeks, continues 30 weeks) can be applied to subject.As needed or specified by health care provider, other (or less) dosage unit can be applied to subject between induction phase and consolidation phase or after consolidation phase.

In other examples, the treatment regimen may include administering a single dosage unit to the subject during the induction phase and administering multiple dosage units during the consolidation phase, wherein multiple dosage units may be administered to the subject by administering dosage units to the subject every 2 weeks. In some cases, a dosage unit is administered to the subject once every 2 weeks. As described above, the consolidation phase may be 30 weeks, so a total of 15 dosage units (e.g., a single dosage unit may be administered once every 2 weeks for 30 weeks) may be administered to the subject. As needed or as prescribed by a health care provider, additional (or less) dosage units may be administered to the subject between the induction phase and the consolidation phase or after the consolidation phase.

In other embodiments, the treatment regimen includes administering a single dosage unit to the subject during the induction phase, administering multiple dosage units during the consolidation phase, and administering multiple dosage units during the maintenance phase. For example, the multiple dosage units during consolidation can be administered to the subject on certain days after the consolidation phase begins. In some cases, the multiple dosage units during consolidation can be administered to the subject by administering more than one dosage unit to the subject at the same time. For example, the multiple dosage units during consolidation can be administered to the subject by administering more than one dosage unit to the subject on a specific day after the consolidation phase begins and administering more than one dosage unit to the subject during the subsequent day during consolidation. The example of this type of treatment regimen can include administering 2 dosage units to the subject after the consolidation phase begins, and administering 2 dosage units on the 43rd day after the consolidation phase begins. Additional (or less) dosage units can be administered to the subject as needed or by healthcare provider regulations between the induction phase and the consolidation phase or after the consolidation phase but before the maintenance phase.

In certain embodiments, multiple dosage units during the maintenance phase can be administered to the subject on certain days after the maintenance phase begins. In some cases, multiple dosage units during the maintenance phase can be administered to the subject by administering more than one dosage unit to the subject at the same time. For example, multiple units during the maintenance phase can be administered to the subject by administering more than one dosage unit to the subject on a specific day after the maintenance phase begins and administering more than one dosage unit to the subject the day after the maintenance phase. An example of this type of treatment regimen may include administering 2 dosage units to the subject on the 2nd day after the maintenance phase begins, and administering 2 dosage units on the 22nd day after the maintenance phase begins. Another example of a treatment regimen during the maintenance phase may include administering 2 dosage units to the subject on the 4th day after the maintenance phase begins, and administering 2 dosage units on the 43rd day after the maintenance phase begins. In some cases, a treatment regimen may include multiple maintenance phases. In some cases, the dosage schedule for each maintenance phase may be the same, or the dosage schedule for other instances during each maintenance phase may be different. Additional (or fewer) dosage units may be administered to the subject between the consolidation phase and the maintenance phase or after the maintenance phase, as needed or as prescribed by a healthcare provider.

In certain embodiments, the dosage unit of the present disclosure can be used before, simultaneously or after other active agents for treating related or unrelated conditions, for example in conjoint therapy. Examples of such additional therapies include radiotherapy, surgical therapy and chemotherapy. If provided simultaneously with other active agents, the dosage unit of the present disclosure can be provided with the same or different preparations. For example, simultaneous treatment can be achieved by administering a dosage unit and a pharmaceutical composition with at least one other active agent, such as a chemotherapeutic agent, which provides a therapeutically effective dose according to a specific treatment regimen combination. The administration of a separate pharmaceutical composition can be carried out simultaneously or at different times (for example, sequentially, in any order, on the same day or on different days), as long as the therapeutically effective effect of the combination of these substances is caused in the subject being treated.

Therefore, aspects of the present disclosure also include combination therapy. In certain embodiments, this method includes administering a therapeutically effective amount of one or more additional active agents. Combination therapy refers to that polyalkylene oxide asparaginase (as described herein) can be used in combination with another therapeutic agent to treat a single disease or condition. In certain embodiments, the compounds of the present disclosure are administered simultaneously with the administration of another therapeutic agent, and can be administered as a component of a composition comprising the compounds of the present disclosure or as a component of a different composition. In certain embodiments, a composition comprising the compounds of the present disclosure is administered before or after the administration of another therapeutic agent.

The subject compound can be used in combination with any medicament that can be used for treating tumor conditions, such as anticancer agents and antitumor agents. A class of interesting anticancer agents includes chemotherapeutics. "Chemotherapy" refers to using one or more chemotherapeutic drugs and/or other medicaments to cancer patients by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, oral or inhaled. The interesting medicament that can be used in combination with the subject compound includes but is not limited to cancer chemotherapeutics, activating agents for reducing cell proliferation, anti-metabolites, microtubule-affecting agents, hormone regulators and steroids, natural products and biological response modifiers. For example, as described in more detail below.

Cancer chemotherapeutics include non-peptide (i.e., non-protein) compounds that reduce cancer cell proliferation, and include cytotoxic agents and cytostatics. Non-limiting examples of chemotherapeutics include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptide compounds may also be used. Suitable cancer chemotherapeutics include dolastatin and its active analogs and derivatives thereof; and auristatin and its active analogs and derivatives thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96/33212, WO 96/14856, and US 6,323,315. For example, dolastatin 10 or auristatin PE may be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include analogs and active analogs and derivatives thereof (see, for example, EP1391213; and Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycin and active analogs and derivatives (including, for example, synthetic analogs, KW-2189 and CB 1-TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepines (PBD)).

The activating agents for reducing cell proliferation are known in the art and are widely used. These activating agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates and triazenes, including but not limited to nitrogen mustards, cyclophosphamide (Cytoxan ^™ ), melphalan (L-hemolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, chlorozotocin, uracil, chloroform, ifosfamide, chlorambucil, pipobroman, triethylene melamine, trivinylthiophosphoramide, busulfan, dacarbazine and temozolomide.

Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, including but not limited to cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-diazine phthalate (PDDF, CB3717), 5,8-diazepam (DDATHF), leucovorin, fludarabine phosphate, pentostatine and gemcitabine.

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, docetaxel, pentostatin, mitomycin-C, azathioprine; brequinar; alkaloids such as vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins such as etoposide, teniposide, etc.; antibiotics such as anthracyclines, daunorubicin hydrochloride (daunorubicin, daunorubicin, idarubicin), idarubicin, dauno ... Bis(R)-1,2-dioxol-1,2-dioxol-2,2-dioxol-3,2-dioxol-4,2-dioxol-5,2-dioxol-6,2-dioxol-7,2-dioxol-8,2-dioxol-9,2-dioxol-1 ,2-dioxol-2 ,2-dioxol-3 ,2-dioxol-4,2-dioxol-8,2-dioxol-9,2-dioxol-1 ,2-dioxol-3 ,2-dioxol-4 ...

Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrozole, capecitabine, reloxafine, cyclophosphamide, ifosfamide, and droloxafine. Microtubule-affecting agents with antiproliferative activity are also suitable and include, but are not limited to, holocolitis (NSC 406042), Halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoctonia solani (NSC 332598), paclitaxel derivatives, docetaxel thiocolchicine (NSC 361792), tritylcysteine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

Suitable hormone modulators and steroids (including synthetic analogs) include, but are not limited to, adrenocortical hormones, such as prednisone, dexamethasone, etc.; estrogens and progestogens, such as hydroxyprogesterone caproate, medroxyprogesterone, megestrol acetate, estradiol, clomiphene citrate, tamoxifen, etc.; and adrenocortical inhibitors, such as aminoglutethimide; 17α-ethinyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, sterol propionate, testosterone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone acetonide, chlorpheniramine, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (flutamide), toremifene (Faloxone) and estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP), carboplatin, etc.; ureas, such as hydroxyurea; and hydrazines, such as N-methylhydrazine; teniposide; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other antiproliferative agents of interest include immunosuppressants, such as mycophenolic acid, thalidomide, desoxaliplatin, azasporine, leflunomide, mizoribine, azaspirane (SKF 105685); (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline), etc.

Taxanes are suitable for use. "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Taxol" (which is understood herein to include analogs, formulations, and derivatives such as, for example, docetaxel, TAXOL ^™ , TAXOTERE ^™ (docetaxel formulations), 10-desacetyl analogs of paclitaxel, and 3'N-desbenzoyl-3'N-tert-butoxycarbonyl analogs of paclitaxel) can be readily prepared using techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. Patents 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 obtained from various commercial sources, including, for example, Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; or T-1912 from Taxus yannanensis). Paclitaxel should be understood to refer not only to the common chemical form of paclitaxel, but also to analogs and derivatives (e.g., Taxotere ^TM docetaxel, as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose). The term "taxane" also includes various known derivatives, including hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazinyl and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288; sulfonamide derivatives described in U.S. Pat. No. 5,821,263; and paclitaxel derivatives described in U.S. Pat. No. 5,415,869. It further includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Applicable biological response modifiers include, but are not limited to, (1) tyrosine kinase (RTK) activity inhibitors; (2) serine/threonine kinase activity inhibitors; (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony stimulating factor; and (9) angiogenesis inhibitors.

Subjects that can be treated using the methods and compositions of the present disclosure include subjects of any age. In some cases, the subject can be an adult. For example, an adult subject can be 18 years of age or older. Subjects that can be treated using the methods and compositions of the present disclosure also include adolescent subjects. For example, a human adolescent subject may be less than 18 years of age. In some cases, the range of the subject is from 1 month to 18 years of age, such as 1 year to 18 years of age, including 2 years of age to 18 years of age, such as 5 years of age to 16 years of age.

In some cases, the method includes that the subject is diagnosed as suffering from AML. Any convenient scheme can be used to diagnose the subject as suffering from AML. In some cases, the French, American and British (FAB) classification systems are used to diagnose and classify acute myeloid leukemia. The diagnosis of acute myeloid leukemia requires that myeloblasts account for 30% (or 20% based on the most recent World Health Organization (WHO) classification system) or more bone marrow cells or circulating white blood cells. The hematological characteristics of the disease define the following various subtypes. The FAB nomenclature (M1 to M7) classifies the subtype of acute myeloid leukemia according to the normal bone marrow elements closest to the outbreak. In some cases, the method includes determining the suitability of the subject diagnosed as suffering from AML to use the polyalkylene oxide asparaginase composition for treatment, for example, as described herein. In some cases, the method includes monitoring the effectiveness of treatment. Any convenient scheme can be used to monitor the effectiveness of treatment.

Preparation method

Aspects of the present disclosure include methods for preparing the polyalkylene oxide asparaginase compositions described herein. In some cases, the method is a method for preparing a liquid polyalkylene oxide-asparaginase composition as described herein. The method can include producing an aqueous composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group, covalently linked to the asparaginase via a linker, a buffer, and a salt.

Embodiments of the method for preparing the polyalkylene oxide-asparaginase composition may include preparing an aqueous concentrate composition. For example, the method for preparing the aqueous concentrate composition may include one or more of the following steps: preparing a solution of asparaginase (e.g., L-asparaginase); attaching a polyalkylene oxide (e.g., polyethylene glycol) to the asparaginase; purifying the polyalkylene oxide-asparaginase; filtering and concentrating the solution of the polyalkylene oxide-asparaginase; diluting the polyalkylene oxide-asparaginase; filtering the polyalkylene oxide-asparaginase and filling the polyalkylene oxide-asparaginase solution into sterile containers; and storing the solution of the polyalkylene oxide-asparaginase.

In the method for producing aqueous concentrate composition, a solution of asparaginase (e.g., L-asparaginase) can be prepared. Asparaginase can be mixed with a solution, such as an aqueous solution (e.g., a buffer solution). Examples of applicable buffers include, but are not limited to, 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, etc., and combinations thereof. In some cases, asparaginase is mixed with a phosphate buffer.

Phosphate buffers can include disodium hydrogen phosphate and sodium dihydrogen phosphate. In some cases, the amount of disodium hydrogen phosphate in the aqueous concentrate composition is 0.05-5 wt.%, such as 0.1-4.5 wt.%, or 0.1-4 wt.%, or 0.1-3.5 wt.%, or 0.1-3 wt.%, or 0.1-2.5 wt.%, or 0.1-2 wt.%, or 0.1-1 wt.%, or 0.1-0.9 wt.%, or 0.1-0.8 wt.%, or 0.1-0.7 wt.%, or 0.1-0.6 wt.%, or 0.2-0.6 wt.%, or 0.3-0.6 wt.%, or 0.4-0.6 wt.%, or 0.5-0.6 wt.%. For example, disodium hydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.1-1 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.2-0.8 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.3-0.6 wt.%. In some cases, disodium hydrogen phosphate can be present in the composition in an amount of 0.5-0.6 wt.%. For example, disodium hydrogen phosphate can 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 sodium dihydrogen phosphate in the aqueous concentrate composition is 0.005-2 wt.%, such as 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 0.0 %. In some cases, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.1-0.4 wt.%, or 0.01-0.2 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, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.05-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.01-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.09-0.15 wt.%. In some cases, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.1-0.2 wt.%. In some cases, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.1-0.15 wt.%. For example, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.12 wt.% (or 0.129 wt.%).

Additional ingredients that can 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 some cases, the salt is sodium chloride.

In some cases, the amount of salt (e.g., sodium chloride) in the aqueous concentrate composition is 0.05-5 wt.%, e.g., 0.05-4 wt.%, or 0.05-3 wt.%, or 0.05-2 wt.%, or 0.1-5 wt.%, or 0.1-4 wt.%, or 0.1-3 wt.%, or 0.1-2 wt.%, or 0.1-1.5 wt.%, or 0.1-1 wt.%, or 0.2-1 wt.%, or 0.3-1 wt.%, or 0.4-1 wt.%, or 0.5-1 wt.%, or 0.6-1 wt.%, or 0.7-1 wt.%, or 0.8-1 wt.%, or 0.8-0.9 wt.%. For example, the salt (e.g., sodium chloride) can be present in the composition in an amount of 0.5-1 wt.%. For example, the salt (e.g., sodium chloride) may be present in the composition in an amount of 0.2-2 wt.%. In some cases, the salt (e.g., sodium chloride) may be present in the composition in an amount of 0.7-1 wt.%. In some cases, the salt (e.g., sodium chloride) may be present in the composition in an amount of 0.8-0.9 wt.%. For example, the salt (e.g., sodium chloride) may be present in the composition in an amount of 0.85 wt.%.

Other aspects of the present disclosure include methods for preparing the freeze-dried, storage-stable compositions described herein. In some cases, the methods are methods for preparing the freeze-dried polyalkylene oxide-asparaginase compositions described herein. The methods can include freeze-drying an aqueous composition comprising a polyalkylene oxide-asparaginase, a buffer, a salt, and a sugar, wherein the polyalkylene oxide-asparaginase has a polyalkylene oxide group covalently linked to the asparaginase via a linker, in a manner sufficient to produce a freeze-dried, storage-stable polyalkylene oxide-asparaginase composition.

In certain embodiments, freeze drying is used to dehydrate the aqueous concentrate composition. In some cases, freeze drying includes removing water from the aqueous concentrate composition. Water can be removed by subliming the water in the composition. For example, the water in the composition can undergo a phase transfer from a solid to a gas. In some cases, freeze drying includes 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 sublimes. During freeze drying, for example, the temperature of the composition drops to below the freezing point of the water in the composition. For example, the temperature of the composition can drop to 0°C or below, or -5°C or below, or -10°C or below, or -15°C or below, or -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 of the composition can drop to -45°C. In some cases, the temperature of the composition may be reduced to -30°C.

In certain embodiments, the pressure around the composition can be reduced to below standard atmospheric pressure.For example, the pressure around the composition can be reduced to 500T or below, for example, 250T or below, or 100T or below, or 50T or below, or 10T or below, or 1T or below, or 500mT or below, or 400mT or below, or 300mT or below, or 200mT or below, or 100mT or below, or 90mT or below, or 80mT or below, or 70mT or below, or 60mT or below, or 50mT or below, or 40mT or below, or 30mT or below, or 20mT or below, or 10mT or below. In some cases, the pressure around the composition can be reduced to 60mT or below, for example, 50mT.

In some embodiments, freeze-drying can also include increasing the temperature of the composition while reducing the pressure around the composition. For example, the temperature of the composition can be increased from a minimum temperature as described above to a temperature higher than the minimum temperature. In some cases, the temperature can be increased to facilitate sublimation of the water in the composition under reduced ambient pressure.

Embodiments of the method for preparing a lyophilized polyalkylene oxide-asparaginase composition can also include producing an aqueous concentrate composition, followed by lyophilization. A process flow diagram for producing the aqueous concentrate composition is shown in FIG1 . As shown in FIG1 , the method for producing the aqueous concentrate composition can include one or more of the following steps: preparing a solution of asparaginase (e.g., L-asparaginase) (10); connecting a polyalkylene oxide (e.g., polyethylene glycol) to the 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 a sterile container (60); and storing the polyalkylene oxide-asparaginase (70) solution.

In the method for producing an aqueous concentrate composition, an asparaginase (e.g., L-asparaginase) solution 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 the like, and combinations thereof. In some cases, the asparaginase is mixed with a phosphate buffer.

In some embodiments, the phosphate buffer can include sodium hydrogen phosphate and sodium dihydrogen phosphate. In some cases, the amount of sodium hydrogen phosphate in the aqueous concentrate composition is 0.05-1 wt.%, such as 0.1-0.9 wt.%, or 0.1-0.8 wt.%, or 0.1-0.7 wt.%, or 0.1-0.6 wt.%, or 0.1-0.5 wt.%, or 0.1-0.4 wt.%, or 0.2-0.4 wt.%, or 0.2-0.3 wt.%, or 0.25-0.3 wt.%. For example, sodium hydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.1-0.5 wt.%. In certain embodiments, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.005-1 wt.%, e.g., 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 0.01-0.2 wt.%, or 0.01-0.1 wt.%, 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, sodium dihydrogen phosphate can be present in the aqueous concentrate composition in an amount of 0.01 to 0.1 wt.

Additional ingredients 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 some cases, the salt is sodium chloride.

In some cases, the amount of salt (e.g., sodium chloride) in the aqueous concentrate composition is 0.05-1 wt.%, e.g., 0.1-0.9 wt.%, or 0.1-0.8 wt.%, or 0.1-0.7 wt.%, or 0.1-0.6 wt.%, or 0.1-0.5 wt.%, or 0.2-0.5 wt.%, or 0.3-0.5 wt.%, or 0.4-0.5 wt.%, or 0.4-0.45 wt.%. For example, the amount of salt (e.g., sodium chloride) in the aqueous concentrate composition is 0.1-1 wt.%.

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

In some cases, the amount of sugar (e.g., sucrose) in the aqueous concentrate composition is 0.1-25 wt.%, e.g., 0.5-20 wt.%, or 1-15 wt.%, or 1-10 wt.%, or 1-9 wt.%, or 1-8 wt.%, or 2-7 wt.%, or 2-6 wt.%, or 3-5 wt.%, or 4-5 wt.%. For example, the sugar (e.g., sucrose) can be present in the aqueous concentrate composition in an amount of 1-10 wt.%.

After the asparaginase solution is prepared, the asparaginase can be connected to a polyalkylene oxide (e.g., polyethylene glycol) so that the polyalkylene oxide is covalently linked to the asparaginase to produce a polyalkylene oxide-asparaginase conjugate. After the polyalkylene oxide-asparaginase is prepared, the solution can be purified. In some cases, the purification comprises filtering the solution through a membrane to remove particulate matter from the solution. The filtration step can produce a substantially purified polyalkylene oxide-asparaginase.

In some cases, the filtered polyalkylene oxide-asparaginase solution is then subjected to a diafiltration and concentration step. The polyalkylene oxide-asparaginase solution can be diafiltered using an ultrafiltration membrane, and the resulting polyalkylene oxide-asparaginase concentrate can be obtained. The concentrate obtained from the diafiltration step can then be diluted so that the solution contains the desired concentration of polyalkylene oxide-asparaginase. Suitable buffers for the dilution step include those described above. In some cases, a phosphate buffer is used to dilute the polyalkylene oxide-asparaginase solution, thereby producing the desired aqueous concentrate composition. For example, the concentrate from the diafiltration step can be diluted so that the resulting aqueous concentrate composition includes an amount of polyalkylene oxide-asparaginase having a potency (activity) 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 some cases, the aqueous concentrate composition includes polyalkylene oxide-asparaginase in an amount of 1,500-3,000 IU/mL. In some cases, the amount of polyalkylene oxide-asparaginase in the aqueous concentrate composition is greater than the amount of polyalkylene oxide-asparaginase in the reconstituted lyophilized composition described herein. In some cases, diafiltration produces substantially purified polyalkylene oxide-asparaginase.

The aqueous concentrate composition can then be filtered and filled into sterile containers. Examples of suitable materials for the container include polymers such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethyl ether (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkane (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), polystyrene, and the like. For example, the container can be a sterile polymer bag. The aqueous concentrate composition can be stored in the container for a certain period of time and can be processed into the freeze-dried, storage-stable composition disclosed herein.

Embodiments of the method may also include transporting the aqueous concentrate composition to a remote location. A "remote location" is a location that is not the location where the aqueous concentrate composition is produced. For example, the remote location may be another location (e.g., an office, a laboratory, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one project is referred to as being "remote" from another project, it means that the two projects may be in the same room, but separated or at least in different rooms or different buildings, and may be at least 1 mile, 10 miles, or 100 miles or more apart.

In certain embodiments, as described above, the method comprises lyophilizing the aqueous concentrate composition in such a manner as to produce a freeze-dried, storage-stable polyalkylene oxide-asparaginase composition. In some cases, lyophilization can be performed in a unit dose container. Lyophilizing the aqueous concentrate composition in a unit dose container to produce a freeze-dried, storage-stable polyalkylene oxide-asparagine composition can facilitate the production of freeze-dried compositions in unit dose containers, for example, by eliminating the need to freeze-dry the aqueous concentrate composition in a separate container and then transfer the freeze-dried composition from the separate container to the unit dose container. As such, in some embodiments, the method comprises introducing the aqueous concentrate composition into a unit dose container and lyophilizing the aqueous concentrate composition in the unit dose container. As described above, the unit dose container can be a vial, for example, a glass vial.

After lyophilization, described method can also include that the lyophilized composition is sealed in unit dose container.For example, sealant or cap can be applied to the opening of unit dose container, thus sealing unit dose container.The time limit of extended can be by sealed container storage, for example 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 (for example 18 months) or more, or 2 years or more, or 2.5 years (for example 30 months) or more, or 3 years or more, or 3.5 years (for example 42 months) or more, or 4 years or more, or 4.5 years (for example 54 months) or more, or 5 years or more.For example, the time limit of extension can be 6 months or more.In some cases, sealed container can be stored 9 months or more.In some cases, sealed container can be stored 1 year (for example 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 (e.g., 24 months) or more.

medicine box

Also provided is a medicine box for implementing the subject method, wherein the medicine box can include one or more of the above-mentioned liquid and/or lyophilized composition.For example, the medicine box can include a unit dose container comprising a liquid composition as described herein.Or, for example, the medicine box can include a unit dose container comprising a lyophilized composition as described herein.In some cases, the medicine box includes two or more unit dose containers, each of which contains a liquid composition as described herein.In some cases, the medicine box includes two or more unit dose containers, each of which contains a lyophilized composition as described herein.In some cases, the medicine box includes two or more unit dose containers, each of which contains a lyophilized composition as described herein.In some cases, the medicine box includes two or more unit dose containers, wherein one or more of the unit dose containers contain a liquid composition as described herein, and one or more of the unit dose containers contain a lyophilized composition as described herein.In certain embodiments, the medicine box includes packaging configured to include unit dose containers.The packaging can be a sealed package, such as a sterile sealed package.Sterile packaging can be configured to seal and isolate the external environment so that there is substantially no microorganism (such as fungi, bacteria, viruses, spore forms, etc.) in the packaging. In some cases, the packaging is optionally sealed in an airtight and/or vacuum seal, such as a water vapor proof package.

In certain embodiments, the medicine box includes a buffer. For example, the medicine box may include a dilution liquid, such as a dilution buffer, which may be suitable for application to a subject, etc. The medicine box may also include other components, such as an applicator, a liquid source, etc., which are applied to implement the subject method. The different components in the medicine box are packaged as needed, such as together or individually. The composition of the subject medicine box may be present in a separate container, or multiple components may be present in a single container, wherein the container and/or packaging (or its part) of the medicine box may be sterile as needed.

Except the composition of raising above, theme medicine box can also comprise the specification sheets of the composition of this medicine box so that implementation theme method.The specification sheets that implement theme method are recorded on applicable recording medium usually.For example, specification sheets can be for example printed on paper or plastic etc.According to this, specification sheets can exist (being attached on packing or sub-packaging) etc. in the form of medicine box packaging insert or its assembly.In other embodiments, specification sheets can exist as electronic storage data file, and it exists on applicable computer readable storage medium, for example portable flash memory, CD-ROM, DVD-ROM, blue disc etc.In other embodiments, actual specification sheets are not present in the medicine box, but provide for example, obtain the guidance of specification sheets from remote source by the Internet.The example of present embodiment is medicine box, and it comprises web address, and wherein specification sheets can be visual and/or can be downloaded from wherein.The same with specification sheets, the present embodiment that obtains specification sheets is recorded on applicable matrix.

use

The subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods are used in applications where it is desired to treat a disease or condition in a subject that is amenable to treatment by administration of polyalkylene oxide-asparaginase. For example, the subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods are used to treat a tumor condition in a subject. In some cases, the subject compositions (e.g., liquid or lyophilized storage-stable compositions) and methods are used to treat cancer in a subject. Examples of cancer types amenable to treatment 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), acute myeloid leukemia (AML), and the like. Thus, the subject lyophilized storage-stable compositions and methods are used to provide therapeutically effective treatments for tumor conditions, such as cancers, including, but not limited to, leukemias, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and the like.

The freeze-dried storage-stable compositions and methods of the present disclosure are applied to treat subjects of any age. In some cases, the subject composition (e.g., liquid or freeze-dried storage-stable composition) and method are applied to treat adults. For example, adults can be 18 years old or above. In other cases, the composition (e.g., liquid or freeze-dried storage-stable composition) and method are applied to treat teenagers. For example, human adolescent subjects can be under 18 years old.

The compositions and methods of the present disclosure are applied to such applications, wherein it is desired that the composition of storage-stable.For example, the compositions and methods of the present disclosure are applied to provide a composition of storage-stable, which is stable (for example, will not significantly degrade and/or retain substantially all of its activity) within the time limit of extension.For example, the compositions and methods of the present disclosure are applied to provide a composition of storage-stable, which can stabilize the time limit of extension, for example, 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 (for example, 18 months) or more, or 2 years or more, or 2.5 years (for example, 30 months) or more, or 3 years or more, or 3.5 years (for example, 42 months) or more, or 4 years or more, or 4.5 years (for example, 54 months) or more, or 5 years or more. In some cases, the compositions and methods of the present disclosure are applied to provide a composition of storage-stable, which can stabilize 9 months or more. In some cases, the compositions and methods of the present disclosure are applied to provide a storage-stable composition, which can be stable for 1 year (e.g., 12 months) or more. In some cases, the compositions and methods of the present disclosure are applied to provide a storage-stable composition, which can be stable for 1.5 years (e.g., 18 months) or more. In some cases, the compositions and methods of the present disclosure are applied to provide a storage-stable composition, which can be stable for 2 years (e.g., 24 months) or more. In certain embodiments, the compositions and methods of the present disclosure are applied to provide a storage-stable composition, which increases 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 present disclosure are used to provide storage-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.

In certain embodiments, the dosage of the present disclosure may be administered before, concurrently with, or after another one or more oncological disorder therapies for the treatment of related or unrelated conditions. If provided concurrently with another oncological disorder therapy, the dosage of the present disclosure may be provided in the same or different formulations. For example, co-therapy may be performed by administering a dosage and pharmaceutical composition with at least another active agent, such as a chemotherapeutic agent, according to a specific treatment regimen, in such a combined manner to provide a therapeutically effective dose. Separate pharmaceutical compositions or treatments may be administered simultaneously or at different times (e.g., sequentially, in any order, on the same day, or on different days), as long as a therapeutically effective dose of these combinations of substances is produced in the therapy being administered to the subject. Thus, the methods and compositions of the present disclosure are used to treat a subject using a combination therapy comprising administering a polyalkylene oxide-asparaginase of the present disclosure with one or more additional active agents and/or therapies (e.g., radiotherapy, chemotherapy, immunotherapy, etc.).

As can be apparent from the disclosure provided above, the embodiments of the present disclosure can have a wide range of applications. Therefore, the embodiments provided herein are provided for illustrative purposes, but it is by no means intended to be considered as limitations to the embodiments of the present disclosure. It is readily appreciated by those skilled in the art that various non-critical parameters can be changed or modified to obtain substantially similar results. Therefore, the following examples are given to provide those skilled in the art with a complete disclosure and description of how to prepare and apply the disclosed embodiments, and are not intended to limit the scope of the present invention to the inventors, nor are they intended to indicate that the following experiments are all or only experiments performed. Attempts have been made to ensure the accuracy (such as amount, temperature, etc.) of the numerical values used, but some experimental errors and deviations should be considered, unless otherwise indicated, parts are given by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

The following examples are provided for illustrative purposes only and are in no way intended to limit the scope of the presently disclosed embodiments. Attempts have been made to ensure accuracy with respect to numbers used (eg, amounts, temperature, etc.), but some experimental errors and deviations should, of course, be allowed for.

Example

Example 1

A concentrated volume of a composition comprising polyethylene glycol-asparaginase with an SS-PEG linker was produced according to the following protocol. After producing the concentrated volume of the composition, a lyophilized composition was produced from the concentrated volume of the composition according to the following protocol. FIG1 shows a process flow diagram for preparing a lyophilized, storage-stable composition according to an embodiment of the present disclosure.

Preparation of L-asparaginase solution

Calculate the amount of L-asparaginase required for processing, weigh it into a 6L stainless steel beaker, and mix for 5-10 minutes. Calculate and weigh the amount of phosphate buffer needed to dilute the L-asparaginase to a target concentration of 5 mg/mL. Then, add the L-asparaginase to the phosphate buffer and mix for 10-15 minutes in a 7-gallon stainless steel pressure vessel. Take a sample and submit it for protein and specific activity testing.

PEGylation

Calculate and weigh the amount of SS-PEG required for this step. Heat the 7-gallon stainless steel pressure vessel containing the diluted L-asparaginase solution to 29-31°C with gentle stirring. Once the L-asparaginase solution reaches the appropriate temperature range, increase the mixer speed and start the dosing pump. Once the pH is adjusted to 7.7-7.9 with 0.5N NaOH, add the SS-PEG to the 7-gallon stainless steel pressure vessel. After 30 minutes, stop the dosing pump and disconnect the temperature jacket.

Purification

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

Diafiltration/concentration

Calculate the amount of PBS required for 15X diafiltration and prepare the diafiltration system. Place a 7-gallon stainless steel pressure vessel on the scale. The Millipore-2 diafiltration system is provided with a membrane with a 100,000Da nominal molecular weight restriction, pre-treated with a diafiltration peristaltic pump and 5L PBS. Then set the liquid level control system, including a buffer peristaltic pump, and place the 7-gallon stainless steel pressure vessel on the scale. The -2 system is filled with polyethylene glycol-asparaginase for adjustment and recirculation for 5 minutes. After adjustment, the permeate waste line is opened and diafiltration is started. After 15 minutes and 30 minutes, samples are obtained from the permeate waste line and loaded into vials to perform activity and protein testing. The material used for lyophilization is diafiltered and then concentrated to 18.0mg/mL or more, with a potency of 1,850IU/mL or more. When diafiltration is complete, the volume of the material in the process in the 7-gallon stainless steel pressure vessel is adjusted to reach the target concentration (18.0mg/mL or more for the process of lyophilized formulations) by removing an appropriate amount of permeate. PBS was then pumped through the system into a 7-gallon stainless steel pressure vessel as a cleaning fluid.

The quality control assays for the diafiltration product were performed, including the determination of the product's impurity profile. Permeate enzyme activity (EEA) was measured after 15 and 30 minutes to ensure that the product was retained by the diafiltration membrane. Free PEG and N-hydroxysuccinimide (NHS) were components of the process-related impurity profile measured in the final product. The normal process controls for the diafiltration unit operation are given in Table 1 below.

The NHS and free-PEG data generated from three drug substance compositions intended for lyophilization (e.g., concentrated volume drug substance compositions) are shown in Table 2. The data generated show that small changes in the diafiltration/concentration process do not affect the quality of the product.

Table 1: Specifications of the bulk drug substance composition intended for lyophilization

Table 2: Batch Analysis of Concentrated Bulk Drug Substance Composition

*Values taken from samples after diafiltration/before dilution

dilution

The in-process material was mixed in a 7-gallon stainless steel pressure vessel and samples were withdrawn for activity and protein measurements. The volume of the in-process material was then diluted with PBS to achieve a protein concentration of ≥18.0 mg/mL (target 20.0 mg/mL), (≥1850 IU/mL activity) for the drug substance of the intended lyophilized composition. The diluted in-process material was then mixed and samples were withdrawn for quality assurance testing (see Table 1).

Sterile filtration

The concentrated solution for lyophilization is filtered at 0.2 μm into disposable 20 L sterile bags for storage until lyophilization is performed. Samples are collected and tested for sterility. The bulk pharmaceutical composition containing polyethylene glycol-asparaginase for lyophilization can be kept in the 20 L bag at 2-8°C for up to 2 months and then lyophilized.

Container sealing

The bulk drug composition containing the polyethylene glycol-asparaginase for freeze drying is processed into a freeze-dried composition after a 0.2 μm filtration step and delivered to a disposable pre-sterilized 20L bioprocessing bag. The construction materials of the bag include low-density polyethylene (LDPE), linear low-density polyethylene (LLDPE), nylon, and ethylene vinyl alcohol (EVOH) layers. The direct product contact surface (inner layer) is LDPE. Each bag has two openings with connecting tubes, which are tightened with closures until it is convenient to receive the bulk drug substance composition including the polyethylene glycol-asparaginase for freeze drying. Based on 25-40 kGy exposure irradiation and release bags, and carry out Limulus Amebocyte Lysate (LAL) endotoxin testing, 100% visible seal visual and gas leakage testing, and 100% visual inspection after assembly. Qualitative testing results showed that the inner layer of the bag passed biological reactivity testing, including USP <88> Class IV plastics testing, USP <87> cytotoxicity testing, and USP <661> physiochemical properties testing. Furthermore, the bag complies with USP <788> for particulate matter for parenteral use, and all extractables testing met the manufacturer's requirements.

Drug substance stability maintenance time

The concentrated bulk drug substance material was monitored at 2-8°C at 0, 2, 4, 6, 8, and 12 weeks. Stability samples were maintained in 250 mL sample bags with a polyethylene product contact surface. Data from the test program are given in Table 3 for Batch 1, Table 4 for Batch 2, and Table 5 for Batch 3.

Stability data for the concentrated drug substance used in the lyophilized composition met the acceptance criteria throughout the 12-week testing program and indicated that the 2-month holding period between concentrated drug substance production and production of the lyophilized composition was acceptable.

Table 3: Stability data of Batch 1 stored at 2-8°C

Table 4: Stability data of Batch 2 stored at 2-8°C

*Values taken from post-diafiltration/pre-dilution samples tested

Table 5: Stability data of batch 3 stored at 2-8°C

Freeze-dried composition

The lyophilized composition powder for injection was produced in a disposable vial containing 3,750 IU of active PEGylated L-asparaginase (750 IU/mL after reconstitution with 5.2 mL WFI). The ingredients of the lyophilized composition after reconstitution included 4.5% sucrose, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride. The composition of the lyophilized composition is provided in Table 6. In addition, the lyophilized composition was provided in a treated glass bottle container (Nipro Type 1) with a 20 mm aluminum flip seal.

Table 6: Compositions of polyethylene glycol-asparaginase compositions

Element grade Amount/g* polyethylene glycol-asparaginase n/a 750IU Disodium hydrogen phosphate USP 2.79mg Sodium dihydrogen phosphate USP 0.60mg Sodium chloride USP 4.25mg sucrose National Formulary (NF) 45mg Water for Injection (WFI) USP Up to 1.0g

*Value reconstructed using WFI

Development of freeze-dried polyethylene glycol-asparaginase formulations

The goal of developing a lyophilized polyethylene glycol-asparaginase formulation was to obtain a stable lyophilized composition that could be stored at 2-8°C or 25°C for at least 18 months.

Initial experiments were conducted to evaluate the feasibility of freeze-dried compositions of polyethylene glycol-asparaginase. Five variations of the freeze-dried compositions were studied. Each formulation contained ~5 mg/mL polyethylene glycol-asparaginase in 50 mM phosphate buffer, pH 6.5, and 5% w/v of one of five different cryoprotectants (mannitol, maltose, sucrose, trehalose, and/or β-HPCD). In addition, polyethylene glycol-asparaginase was freeze-dried without a cryoprotectant. These six freeze-dried compositions were compared with the batch numbers of polyethylene glycol-asparaginase in the liquid formulations used to prepare these different formulations. The SEC-purity (GF-HPLC) method was used to evaluate the quality of the polyethylene glycol-asparaginase compositions. The results of the study are presented in Table 7. As can be seen from Table 7, sucrose was the most effective in preserving the purity of the polyethylene glycol-asparaginase during the freeze-drying process. The purity of the lyophilized composition containing 5% sucrose (83.0%) was comparable to the purity of the liquid polyethylene glycol-asparaginase formulation used to prepare the six formulations for subsequent lyophilization (83.8%).

Table 7: Feasibility study results for lyophilizing polyethylene glycol-asparaginase

^1All formulations also contain ~5 mL of pegaspargase injection in 5 mM phosphate buffer, pH 6.5

^2Expected retention time of pegaspargase injection

Several different excipients (e.g., sucrose, trehalose, mannitol, polysorbate 80) were evaluated as potential stabilizers for inclusion in lyophilized compositions. Four experiments were conducted, each including four different compositions. Small batches of lyophilized polyethylene glycol-asparaginase were prepared using the different excipients and their stability was evaluated. A list of the formulations tested is shown in Table 8.

Table 8: Polyethylene glycol-asparaginase compositions tested

All batches described in Table 8 were evaluated in stability testing at 5°C, 25°C, and 40°C and were evaluated using a battery of tests (activity, specific activity, protein, pH, purity (GF-HPLC), aggregates (GF-HPLC), and particulate matter) that evaluate the key quality attributes and stability of polyethylene glycol-asparaginase. Based on the analysis of stability data collected from the 16 different formulations described in Table 8, sucrose was identified as a suitable cryoprotectant (e.g., stabilizer).

Various sucrose concentrations were measured to assess the concentrations that were suitable for more stable (i.e., the freeze-drying scheme that had less impact) and more stable products. Other small batches containing different concentrations of sucrose were prepared, as summarized in Table 9. The sucrose and PEG-asparaginase concentrations shown in Table 9 show the amounts present in the concentrated raw drug substance. During the freeze-drying process, before starting freeze-drying, the vials were filled at 2.5 mL. These freeze-dried vials were reconstructed with 5.0 mL to obtain final sucrose and PEG-asparaginase concentrations, which were approximately half the concentrations shown in the table. This study shows that the increase in sugar content allows for higher freeze-drying temperatures, which puts less pressure on the freeze-dried product and also reduces the entire freeze-drying cycle time and produces a drier (more stable) freeze-dried product.

Table 9: Sucrose compositions tested

1Concentration during lyophilization: ^2.5 mL/vial. Concentration is reduced by half after reconstitution of the final product with 5 mL/vial of WFI.

The five batches described in Table 9 were also evaluated in stability testing at 5°C, 25°C and 40°C. Quality attributes such as activity, specific activity, protein, pH, purity (GF-HPLC), aggregates (GF-HPLC) and particles were evaluated during this stability study. The purity and potency obtained for the accelerated (25°C) and loaded (40°C) stability studies for these batches are shown in Figures 2-5. These stability data indicate that the formulation containing 10% sucrose (5% sucrose after reconstitution of the final product with 5 mL/vial WFI) provides a product with the best stability in purity and potency. Other quality attributes were less affected by the different formulations and were stable in the batches that also contained 10% sucrose. Figure 2 shows a graph of purity (%) versus time (weeks) at 40°C for freeze-dried PEG-asparaginase compositions of batches 5A, 5B, 5C, 5D, 5E and 1A. Figure 3 shows a graph of the potency (IU/mL) versus time (weeks) for lyophilized PEG-asparaginase compositions of Lots 5C, 5D, 5E, and 1A at 40°C. Figure 4 shows a graph of the purity (%) versus time (weeks) for lyophilized PEG-asparaginase compositions of Lots 5A, 5B, 5C, 5D, 5E, and 1A at 25°C. Figure 5 shows a graph of the potency (IU/mL) versus time (weeks) for lyophilized PEG-asparaginase compositions of Lots 5C, 5D, 5E, and 1A at 25°C.

Freeze-dried

Prepare a buffer solution consisting of water for injection (WFI), disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and sucrose. The buffer is used to dilute the concentrated bulk drug substance.

WFI is added to a beaker at 90% of the target weight required to prepare the buffer solution. Then, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride are weighed separately and added to the WFI and mixed until dissolved. The required amount of sucrose is weighed and added to the mixed buffer solution and mixed to dissolve. The pH of the solution is then measured and slowly adjusted to 7.3±0.1 by adding NaOH. WFI is added to the buffer solution as needed, and the volume of concentrated bulk solution required for the batch size is weighed using the density of the concentrated bulk solution. Buffer samples are collected for sucrose determination. The concentrated bulk solution is added to the buffer solution, and the final solution is mixed for not less than (NLT) 10 minutes. After mixing is complete, a confirmed pH measurement (7.3±0.1) is obtained from the top, middle, and bottom of the container, and samples are collected for testing during protein, density, sucrose, and pre-filtration bioburden.

After final formulation, the solution was sterile filtered. The previously sterilized filter tube assembly was placed in the formulation bulk container and then filtered through two 0.22 μm filters located on the filter tube assembly into a pre-sterilized 10 L bioprocess bag in preparation for filling. After transferring the entire product to the 10 L bioprocess bag, the filter tube assembly was separated from the bioprocess bag and the filter integrity was tested. A 20 mL pre-lyophilized sample was collected from the upstream filter (unfiltered) and each finished drug product specification test.

A flow chart illustrating the final formulation and sterile filtration process steps is shown in FIG6 .

Sterile filtration and lyophilization

After sterile filtration was completed, a 10 L biobag containing the volume of API solution was connected to the fill tube assembly and the product vials were then filled and partially stoppered using a Flexicon FMB210 filter at a target fill weight of 2.5 g/vial. The fill weight was monitored by performing a minimum of one weight check (1 vial) for each tray filled (action limit: 2.43-2.57 g, alarm limit: 2.38-2.62 g) during the filling operation. When the filling operation was complete, 20 pre-lyophilized vials were tested and all remaining filled vials were transferred to stainless steel lyophilization trays and subsequently loaded into a pre-cooled (5°C) 270 ^ft2 Hull freeze drying system for lyophilization. The lyophilization process included the stages shown in Table 10.

Table 10: Freezing cycle - process parameters

After the lyophilization cycle is complete, the vacuum chamber is backfilled with nitrogen and the vials are fully stoppered. Figure 7 shows a flow chart of the aseptic filling and lyophilization process steps. The fully stoppered vials are then packaged into cartons (90 vials/carton) for storage and/or shipping.

Quality standards for freeze-dried compositions

The product quality standards of the lyophilized composition are shown in Table 11.

Table 11: Quality standards of freeze-dried compositions

^1Except for pre-reconstruction appearance tests, all tests were performed after reconstruction using WFI;

Batch analysis

Batch analysis for the lyophilized composition product batches is shown below in Table 12. The results for all three batches were within published specifications.

Table 12: Batch analysis of lyophilized compositions

Stability studies

Lyophilized drug product batches 1, 2, and 3 were subjected to long-term (2-8°C) and accelerated (25 ± 3°C; 60% ± 5% RH) stability testing. These batches were also subjected to a heat stress stability test (40 ± 2°C; 75% ± 5% RH) to evaluate the heat-induced degradation characteristics of the product.

Long-term stability (2-8°C)

Stability data for batches of lyophilized drug products stored under long-term (2-8°C) conditions are provided in Tables 13-16. The long-term stability data indicate that the lyophilized drug products stored at 5±3°C remained within the acceptance criteria for all stability time points. Water content (KF) data ranged from 0.96% to 1.35% (quality standard = NMT 3.0%) up to 12 weeks under 2-8°C storage conditions. Unlike commercial liquid drug products, which show an increase in activity and a decrease in purity over time, this trend was not observed for the lyophilized compositions. Schematic diagrams of the stability of purity (Figure 8) and potency (Figure 9) as well as aggregates (Figure 10) at 2-8°C are shown in the accompanying drawings.

Table 13: Long-term stability data of batch 1

1Tested at ⁹ months using MilliQ water used to reconstitute the drug product.

Table 14: Long-term stability data of batch 2

1Tested at ⁹ months using MilliQ water used to reconstitute the drug product.

Table 15: Long-term stability data of batch 3 (upright)

1Tested at ⁹ months using MilIiQ water for reconstitution of drug product.

Table 16: Long-term stability data of batch 3 (inverted)

Accelerated stability (25±3℃; 60%±5%RH)

Stability data for the lyophilized drug product batches stored under accelerated conditions (25 ± 3°C; 60% ± 5% RH) are shown in Tables 17-20. The stability data demonstrate that the lyophilized drug product stored under accelerated conditions remained well within acceptance criteria at all stability time points. The moisture content (KF) at 25°C was 1.12%-1.23% (quality specification = NMT 3.0%) at 4 weeks. Graphs of the stability of the quality attributes purity ( FIG11 ) and potency ( FIG12 ), as well as aggregates ( FIG13 ), at 25 ± 3°C are shown in the accompanying figures.

Table 17: Accelerated Stability Data for Batch 1

Table 18: Batch 2 Accelerated Stability Data

Table 19: Batch 3 (upright) accelerated stability data

Table 20: Batch 3 (Inverted) Accelerated Stability Data

Thermal load stability (40±2℃; 75%±5%RH)

Tables 21-24 provide stability data for batches of the lyophilized drug product stored under stress (40 ± 2°C; 75% ± 5% RH). The stability data demonstrate that the lyophilized drug product stored under stress remained within acceptance criteria throughout the study period. Water content (KF) ranged from 1.16% to 1.45% (quality specification = NMT 3.0%) up to 4 weeks of storage at 40°C. Graphs of potency ( FIG. 14 ) and purity ( FIG. 15 ) at 40 ± 2°C, as well as aggregate stability ( FIG. 16 ), are provided in the accompanying figures.

Table 21: Heat load stability data for batch 1

Table 22: Heat load stability data for batch 2

Table 23: Batch 3 (upright) heat load stability data

Table 24: Batch 3 (Inverted) Heat Load Stability Data

Example 2

A composition comprising polyethylene glycol-asparaginase with a SC-PEG linker (i.e., a succinimidyl carbonate linker) was produced to yield a long-acting polyethylene glycol asparaginase (succinimidyl carbonate-polyethylene glycol [SC-PEG] E. coli L-asparaginase). The composition of this composition is provided in Table 25.

Table 25: Components of Calaspargase Pegol Composition

Element grade Amount/g* Long-acting polyethylene glycol asparaginase n/a 750IU Disodium hydrogen phosphate USP 5.58mg Sodium dihydrogen phosphate USP 1.29mg Sodium chloride USP 8.50mg Water for Injection (WFI) USP Up to 1.0g

Example 3

After producing the composition of concentrated volume, a lyophilized long-acting polyethylene glycol asparagine composition can be produced from the composition of concentrated volume. Fig. 1 shows an example of a process flow diagram for preparing a lyophilized storage-stable composition according to an embodiment of the present disclosure. The lyophilized composition powder for injection can be produced in a disposable vial containing 3,750 IU of active long-acting polyethylene glycol asparagine (750 IU/mL after reconstitution with 5.2 mL of WFI). The ingredients of the lyophilized composition may include 4.5% sucrose, disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride after reconstitution. The composition of the lyophilized composition is provided in Table 26.

Table 26: Ingredients of long-acting polyethylene glycol asparaginase composition

* Value reconstructed using WFI

Example 4

The purpose of this study was to provide comparative pharmacokinetic (PK), pharmacodynamic (PD) and immunogenicity information for liquid pegaspargase (PEG-L-asparaginase;) and lyophilized pegaspargase, administered intravenously, by slow push injection, once (day 1), or weekly for 4 weeks (days 1, 15, 22, 29, and 36) to beagle dogs. Because reconstituted lyophilized pegaspargase is administered intravenously to humans, the same route of administration was used in this study. Single-dose and repeated-dose PK/PD studies are essential to determine and compare the pharmacokinetics and pharmacodynamics of equivalent doses of liquid and reconstituted lyophilized forms.

Beagle dogs (nominally 5/sex/group) were given 500 IU/kg of liquid pegaspargase or reconstituted lyophilized pegaspargase by intravenous injection in a dose volume of 0.667 mL/kg (see Table 27). Beagle dogs were approximately 6 months old, males weighing 7.2 kg to 10.7 kg, and females weighing 5.6 kg to 8.2 kg.

Liquid pegaspargase (5 mL PBS buffer containing 50 mM phosphate and 0.85% saline at pH 7.2-7.4) was used as provided and did not require preparation. Lyophilized pegaspargase (see Example 1) was prepared for administration by aseptically reconstituting the contents of a vial with 5.2 mL water for injection (WFI) (using a No. 21 syringe) to a concentration of 750 IU/mL. The contents of the vial were gently swirled until completely dissolved. The mixture was visually inspected for particulate matter, turbidity, or discoloration prior to administration. A fresh formulation was prepared for each day of dosing, maintained at room temperature, and used within 2 hours of preparation.

Table 27: Regimens and Dosing Schedule

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 were given repeated doses of liquid pegaspargase or lyophilized pegaspargase, respectively. The test article was administered intravenously as a slow push (over approximately 2 minutes) within 2 hours. An indwelling catheter (non-butterfly catheter) was used, followed by a saline flush to clear the catheter cap of any remaining dose volume. A straight needle was inserted into the catheter cap to ensure consistent needle placement within 2 minutes.

Blood samples were obtained from all animals on day 1 and day 36 for pharmacokinetic and pharmacodynamic analysis. Approximately 1.0 mL of whole blood was obtained at each time point. Prior to blood collection, animals were unanesthetized and non-fasted. Blood was collected in tubes containing sodium heparin anticoagulant and placed in an upright position on wet ice. Within 5 minutes of blood collection, the blood samples were centrifuged for 5 minutes (at approximately 3000 rpm, approximately 4°C) to obtain plasma.

PD: Pipette a 125 μL aliquot of plasma into a frozen tube prefilled with 125 μL of SeraPrep for asparagine determination. Invert the tube three times to mix the SeraPrep and immediately flash freeze in liquid nitrogen or methanol/dry ice within 15 minutes of blood sample collection. Analyze all aliquots containing SeraPrep by high-performance liquid chromatography (HPLC) with mass spectrometry detection (LC-MS/MS) for asparagine determination.

PK: The remaining plasma was divided into two cryovials for asparaginase activity assay and snap-frozen within 30 minutes of blood sample collection. All aliquots without SeraPrep were assayed for asparaginase activity by a colorimetric mixed enzyme reaction.

result

Analysis performed during lyophilized pegaspargase treatment confirmed that the appropriate concentration of the dose formulation was administered (expected protein concentration of 6.6 mg/mL and 741 IU/mL activity per batch analyzed) (Table 28).

Table 28: Analytical Chemistry

The results of the pooled group mean plasma concentrations (Cmax) of asparaginase and the pooled area under the plasma asparaginase concentration-time curve estimated to 552 hours after the dose on Day 1 and after repeat dosing on Day 36 ( _AUC0-552 ) for each group (sex combined) are summarized in Table 29.

Table 29: Bioequivalence: Pooled Group Mean Data (Combined Genders)

The study design was a parallel group design and the data were statistically analyzed using analysis of variance techniques. The Cmax and AUC _0-552 data from both days were analyzed using an ANOVA model with formulation, time, sex, and their interaction as factors.

The two pegaspargase preparations were analyzed for Cmax and AUC _0-552 , and the corresponding two-sided 90% CI for the ratio of geometric means are summarized in Table 30.

Table 30: Pharmacokinetic data

For Cmax, there was evidence of bioequivalence because the confidence interval (0.867 to 0.970) was contained within the critical region.For _AUC0-552 , there was evidence of bioequivalence because the confidence interval (0.841-0.996) was contained within the critical region.

The mean maximum plasma concentrations of pegaspargase (Cmax) and the mean area under the plasma pegaspargase concentration-time curve estimated up to 552 hours after the Day 1 dose and after repeat dosing on Day 36 ( _AUC0-552 ) are summarized below with standard deviations in parentheses in Table 31 (by sex).

Table 31: Plasma concentration-time characteristics

The correlation between the mean maximum plasma concentration of asparaginase (Cmax), the mean area under the plasma asparaginase concentration-time curve (AUC _0-552 ), and the ratios for the dose levels of the lyophilized formulation compared to the liquid formulation are shown in Table 32.

Table 32: Correlation between mean Cmax, mean AUC and dose levels

Systemic exposure to asparaginase in dogs was similar in Cmax and AUC _0-552 values following administration of the reconstituted lyophilized pegaspargase compared to the liquid formulation following repeated dosing on Days 1 and 36. There was no evidence of significant differences between formulations for AUC _0-552 , but there was some evidence of significant differences between formulations for Cmax values, with Cmax being slightly lower (8%) after administration of the lyophilized product compared to the liquid formulation.

Systemic exposure Cmax and _AUC0-552 values for asparaginase in female dogs were generally similar to those for males, and there was no evidence of any statistically significant differences in systemic exposure at Cmax or _AUC0-552 .

Other parameters assessed during the study were: vitality, clinical observations, body weight, food consumption, respiratory rate, body temperature, hematology, coagulation, and blood chemistry; no adverse effects related to the test article were observed in vivo.

After repeated intravenous doses (day 36), the Cmax values and extent of systemic exposure of dogs to asparaginase (AUC _0-552 ) were higher than those after a single dose (day 1), and these differences were statistically significant (p < 0.001). The mean accumulation ratio calculated based on the AUC _0-552 values (note that data from different animals for each day were provided) was greater than 1, indicating that accumulation of asparaginase occurred after repeated intravenous administration of liquid pegaspargase.

In summary, the two pegaspargase formulations demonstrated equivalence in Cmax and AUC _0-552 , as the ratio of the corresponding two-sided 90% CI geometric means fell well within the conventional margin of bioequivalence range of 0.8-1.25. There were no significant differences in AUC _0-552 between the two formulations. Systemic exposure to asparaginase was similar with both products, and some accumulation occurred in both sexes with repeated dosing. Asparaginase was fully inhibited in all animals for up to 336 hours and in the majority of animals for up to 552 hours.

In conclusion, there were no significant differences between liquid pegaspargase or reconstituted lyophilized pegaspargase at a dose of 500 IU/kg, and they had comparable pharmacokinetic, pharmacodynamic, and immunogenic properties.

Implementation Plan

In one embodiment, the present disclosure provides a lyophilized storage-stable composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently linked to an asparaginase via a linker. The lyophilized storage-stable composition further comprises a buffer, a salt, and a sugar.

In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight of 2,000-10,000 Daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 Daltons.

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 succinate linker.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 500-1,000 IU/g.

In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, disodium hydrogen phosphate is present in an amount of 0.1-0.5 wt.%. In some embodiments, sodium dihydrogen phosphate is present in an amount of 0.01-0.1 wt.%.

In some embodiments, the salt is sodium chloride.In some embodiments, the sodium chloride is present in an amount of 0.1-1 wt.%.

In some embodiments, the sugar comprises a disaccharide. In some embodiments, the disaccharide comprises sucrose. In some embodiments, the sugar comprises sucrose in an amount of 1-10 wt.%.

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

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol having a molecular weight of 5,000 Daltons, the asparaginase is Escherichia coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising 0.25-0.3 wt.% disodium hydrogen phosphate and 0.05-0.07 wt.% sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol 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 0.279 wt.% disodium hydrogen phosphate and 0.06 wt.% sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 0.425 wt.%, and the sugar is sucrose in an amount of 4.5 wt.%.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol having a molecular weight of 5,000 Daltons, the asparaginase is E. coli asparaginase, the linker is a succinate linker, the buffer is a phosphate buffer comprising 0.25-0.3 wt.% disodium hydrogen phosphate and 0.05-0.07 wt.% sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol having a molecular weight of 5,000 Daltons, the asparaginase is E. coli asparaginase, the linker is a succinate linker, the buffer is a phosphate buffer comprising 0.279 wt.% disodium hydrogen phosphate and 0.06 wt.% sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 0.425 wt.%, and the sugar is sucrose in an amount of 4.5 wt.%.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol 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 0.558 wt.% disodium hydrogen phosphate and 0.129 wt.% sodium dihydrogen phosphate, and the salt is sodium chloride in an amount of 0.85 wt.%.

In another embodiment, the present disclosure provides a lyophilized storage-stable composition comprising a polyethylene glycol-asparaginase having a polyethylene glycol group covalently linked to an E. coli asparaginase via a succinate linker. In another embodiment, the present disclosure provides a lyophilized storage-stable composition comprising a polyethylene glycol-asparaginase having a polyethylene glycol group covalently linked to an E. coli asparaginase via a urethane linker. The lyophilized storage-stable composition further comprises a phosphate buffer, a salt, and an optional disaccharide as summarized in the above embodiments.

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

In some embodiments, the method comprises reconstitution of a lyophilized, storage-stable composition of the present disclosure, producing a reconstituted dosage unit, and administering the reconstituted dosage unit to a subject to deaminize asparagine in the subject. In some embodiments, the reconstitution comprises combining the lyophilized, storage-stable composition with water for injection (WFI).

In some embodiments, the dosage unit comprises 700-800 IU/mL of polyalkylene oxide-asparaginase.

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

In some embodiments, the dosage unit comprises 0.45-1.5 mg/g sodium dihydrogen phosphate. In some embodiments, the dosage unit comprises 0.45-0.75 mg/g sodium dihydrogen phosphate. In some embodiments, the dosage unit comprises 1-2 mg/g sodium dihydrogen phosphate. In some embodiments, the dosage unit comprises 1-1.5 mg/g sodium dihydrogen phosphate.

In some embodiments, the dosage unit comprises 4-9 mg/g sodium chloride. In some embodiments, the dosage unit comprises 4-4.5 mg/g sodium chloride. In some embodiments, the dosage unit comprises 8-9 mg/g sodium chloride.

In some embodiments, the dosage unit includes sucrose. In some embodiments, sucrose is present in an amount of 40-50 mg/g.

In some embodiments, the reconstituted dosage unit delivers 1,500-3,000 IU/m ² of polyalkylene oxide-asparaginase to the subject. In some embodiments, the reconstituted dosage unit delivers 2,000-2,750 IU/m ² of polyalkylene oxide-asparaginase to the subject.

In some embodiments, the method is a method of treating an oncological condition in a subject. In some embodiments, the oncological 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).

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 to the subject 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 3 weeks. In some embodiments, the plurality of reconstituted dosage units is administered to the subject by administering a reconstituted dosage unit to the subject every 2 weeks.

In some embodiments, the subject is an adolescent. In some embodiments, the subject is an adult.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol 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 mg/g disodium hydrogen phosphate and 0.5-0.7 mg/g sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 4-4.5 mg/g, and the sugar is sucrose in an amount of 40-50 mg/g.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol 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 disodium hydrogen phosphate and 0.6 mg/g sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 4.25 mg/g, and the sugar is sucrose in an amount of 45 mg/g.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol having a molecular weight of 5,000 Daltons, the asparaginase is E. coli asparaginase, the linker is a succinate linker, the buffer is a phosphate buffer comprising 2.5-3 mg/g disodium hydrogen phosphate and 0.5-0.7 mg/g sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 4-4.5 mg/g, and the sugar is sucrose in an amount of 40-50 mg/g.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 750 IU/mL, the polyalkylene oxide group comprises polyethylene glycol having a molecular weight of 5,000 Daltons, the asparaginase is E. coli asparaginase, the linker is a succinate linker, the buffer is a phosphate buffer comprising 2.79 mg/g disodium hydrogen phosphate and 0.6 mg/g sodium dihydrogen phosphate, the salt is sodium chloride in an amount of 4.25 mg/g, and the sugar is sucrose in an amount of 45 mg/g.

In another embodiment, the present disclosure provides a method for preparing a lyophilized polyalkylene oxide-asparaginase composition by lyophilizing an aqueous concentrate composition in a manner sufficient to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. The aqueous concentrate composition comprises a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently linked to the asparaginase via a linker, a buffer, a salt, and a sugar.

In some embodiments, the aqueous concentrate composition includes 1,500-3,000 IU/mL of polyalkylene oxide-asparaginase.

In some embodiments, the aqueous concentrate composition includes 0.1-0.5 wt. % disodium hydrogen phosphate.

In some embodiments, the aqueous concentrate composition includes 0.01-0.1 wt. % sodium dihydrogen phosphate.

In some embodiments, the aqueous concentrate composition includes 0.1-1 wt. % sodium chloride.

In some embodiments, the aqueous concentrate composition includes sucrose. In some embodiments, the sucrose is present in an amount of 1-10 wt.%. In some embodiments, the method further comprises producing the aqueous concentrate composition.

In some embodiments, the method further comprises introducing the aqueous concentrate composition into a unit dose container and lyophilizing the aqueous concentrate 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 further comprises sealing the lyophilized composition in the unit dose container.

In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight of 2,000-10,000 Daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 Daltons.

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 succinate linker.

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 Escherichia coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising disodium hydrogen phosphate in an amount of 0.25-0.3 wt.% and sodium dihydrogen phosphate in an amount of 0.05-0.07 wt.%, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

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 succinate linker, the buffer is a phosphate buffer comprising disodium hydrogen phosphate in an amount of 0.25-0.3 wt.% and sodium dihydrogen phosphate in an amount of 0.05-0.07 wt.%, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

In another embodiment, the present disclosure provides a kit comprising two or more unit dose containers, each containing a lyophilized, storage-stable composition comprising a polyalkylene oxide-asparaginase having a polyalkylene oxide group covalently linked to the asparaginase via a linker, a buffer, a salt, and a sugar.

In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight of 2,000-10,000 Daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 Daltons.

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 succinate linker.

In some embodiments, the polyalkylene oxide-asparaginase is present in an amount of 500-1,000 IU/g.

In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, disodium hydrogen phosphate is present in an amount of 0.1-0.5 wt. %. In some embodiments, sodium dihydrogen phosphate is present in an amount of 0.01-0.1 wt. %.

In some embodiments, the salt is sodium chloride.In some embodiments, the sodium chloride is present in an amount of 0.1-1 wt.%.

In some embodiments, the sugar comprises a disaccharide. In some embodiments, the disaccharide comprises sucrose. In some embodiments, the sugar comprises sucrose present in an amount of 1-10 wt.%.

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

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 Escherichia coli asparaginase, the linker is a urethane linker, the buffer is a phosphate buffer comprising disodium hydrogen phosphate in an amount of 0.25-0.3 wt.% and sodium dihydrogen phosphate in an amount of 0.05-0.07 wt.%, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

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 succinate linker, the buffer is a phosphate buffer comprising disodium hydrogen phosphate in an amount of 0.25-0.3 wt.% and sodium dihydrogen phosphate in an amount of 0.05-0.07 wt.%, the salt is sodium chloride in an amount of 0.4-0.45 wt.%, and the sugar is sucrose in an amount of 4-5 wt.%.

In another embodiment, the present disclosure provides a method for treating acute myeloid leukemia (AML) in a subject. The method comprises administering to the subject a dose of polyalkylene oxide-asparagine effective to treat the subject's AML, wherein the polyalkylene oxide-asparaginase has a polyalkylene oxide group covalently linked to the asparaginase via a linker.

In some embodiments, the polyalkylene oxide group comprises a polyethylene glycol group. In some embodiments, the polyethylene glycol group has a molecular weight of 2,000-10,000 Daltons. In some embodiments, the polyethylene glycol group has a molecular weight of 5,000 Daltons.

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 succinate linker.

In some embodiments, the dose comprises 700-800 IU/mL of polyalkylene oxide-asparaginase.

In some embodiments, the dosage comprises a buffer and a salt. In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the dosage comprises 5.25-5.75 mg/g disodium hydrogen phosphate. In some embodiments, the dosage comprises 1.0-1.5 mg/g sodium dihydrogen phosphate. In some embodiments, the salt is sodium chloride. In some embodiments, the dosage comprises 8-9 mg/g sodium chloride.

In some embodiments, the dose comprises 750 IU/mL 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 is a phosphate buffer comprising 5.25-5.75 mg/g disodium hydrogen phosphate and 1-1.5 mg/g sodium dihydrogen phosphate, and the salt comprises 8-9 mg/g sodium chloride.

In some embodiments, the dose comprises 750 IU/mL 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 is a phosphate buffer comprising 5.58 mg/g disodium hydrogen phosphate and 1.29 mg/g sodium dihydrogen phosphate, and the salt comprises 8.5 mg/g sodium chloride.

In some embodiments, the dose comprises 750 IU/mL 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 succinate linker, the buffer is a phosphate buffer comprising 5.25-5.75 mg/g disodium hydrogen phosphate and 1-1.5 mg/g sodium dihydrogen phosphate, and the salt comprises 8-9 mg/g sodium chloride.

In some embodiments, the dose comprises 750 IU/mL 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 succinate linker, the buffer is a phosphate buffer comprising 5.58 mg/g disodium hydrogen phosphate and 1.29 mg/g sodium dihydrogen phosphate, and the salt comprises 8.5 mg/g sodium chloride.

In some embodiments, the dosage comprises a buffer, a salt, and a sugar. In some embodiments, the buffer comprises a phosphate buffer. In some embodiments, the phosphate buffer comprises disodium hydrogen phosphate and sodium dihydrogen phosphate. In some embodiments, the dosage comprises 2.5-3 mg/g disodium hydrogen phosphate. In some embodiments, the dosage comprises 0.45-0.75 mg/g sodium dihydrogen 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 sugar comprises a disaccharide. In some embodiments, the disaccharide comprises sucrose. In some embodiments, sucrose is present in an amount of 40-50 mg/g.

In some embodiments, the dose comprises 750 IU/mL 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 is a phosphate buffer comprising 2.5-3 mg/g disodium hydrogen phosphate and 0.5-0.7 mg/g sodium dihydrogen phosphate, the salt comprises 4-4.5 mg/g sodium chloride, and the sugar comprises 40-50 mg/g sucrose.

In some embodiments, the dose comprises 750 IU/mL 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 is a phosphate buffer comprising 2.79 mg/g disodium hydrogen phosphate and 0.6 mg/g sodium dihydrogen phosphate, the salt comprises 4.25 mg/g sodium chloride, and the sugar comprises 45 mg/g sucrose.

In some embodiments, the dose comprises 750 IU/mL 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 succinate linker, the buffer is a phosphate buffer comprising 2.5-3 mg/g disodium hydrogen phosphate and 0.5-0.7 mg/g sodium dihydrogen phosphate, the salt comprises 4-4.5 mg/g sodium chloride, and the sugar comprises 40-50 mg/g sucrose.

In some embodiments, the dose comprises 750 IU/mL 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 succinate linker, the buffer is a phosphate buffer comprising 2.79 mg/g disodium hydrogen phosphate and 0.6 mg/g sodium dihydrogen phosphate, the salt comprises 4.25 mg/g sodium chloride, and the sugar comprises 45 mg/g sucrose.

In some embodiments, the method further comprises producing the dose by reconstituting the lyophilized storage-stable composition.

In some embodiments, the dose delivers 1,500-3,000 IU/m ² of polyalkylene oxide-asparaginase to the subject. In some embodiments, the dose delivers 2,000-2,750 IU/m ² of polyalkylene oxide-asparaginase to the subject.

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 dose to the subject during the induction phase and administering multiple doses to the subject during the maintenance phase. In some embodiments, the multiple doses are administered to the subject by administering a dose to the subject every 3 weeks. In some embodiments, the multiple doses are administered to the subject by administering a dose to the subject every 2 weeks.

In some embodiments, the subject is an adolescent. In some embodiments, the subject is an adult.

Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Therefore, the above only illustrates the principles of the embodiments of the present disclosure. It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in this article, embody the principles of the embodiments of the present invention in their spirit and scope. In addition, all embodiments and conditional language described herein are mainly for understanding the principles of the embodiments of the present disclosure and the concepts provided by the inventors for further fields, and should be interpreted as not being limited to the embodiments and conditions of these specific descriptions. In addition, all statements herein describing the embodiments of the present disclosure, aspects and reality are expected to cover their structural and functional equivalents. In addition, it is expected that such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, any elements that perform the same functional development, regardless of structure. Therefore, the scope of the embodiments of the present disclosure is not intended to be limited to the embodiments shown and described herein. On the contrary, the scope and spirit of the embodiments of the present disclosure are embodied by the appended claims.

Claims (28)

1. A reconstructed lyophilized composition comprising: Polyalkylene oxide-asparaginase, at a concentration of 100 to 5000 IU per milliliter of the composition; Disodium hydrogen phosphate at a concentration of 0.1 to 0.8 wt.%; Sodium dihydrogen phosphate with a concentration of 0.01 to 0.5 wt.%; The salt concentration based on the weight of the composition is 0.05-1 wt.%; and The sugar in the composition has a concentration of 0.1 to 25 wt.% by weight, wherein the polyalkylene oxide-asparaginase comprises an asparaginase covalently linked to a polyalkylene oxide group, and wherein the sugar is sucrose. The polyalkylene oxide group includes a polyethylene glycol group, and the salt is sodium chloride. 2. The composition of claim 1, wherein the concentration of said salt is 0.1 to 1 wt.%, 0.2 to 0.5 wt.%, or 0.3 to 0.5 wt.% based on the weight of said composition. 3. The composition of claim 1, wherein the concentration of said sugar is 0.5-20 wt.%, 1 to 10 wt.%, or 4 to 5 wt.% based on the weight of said composition. 4. The composition according to claim 1, comprising: Polyalkylene oxide-asparaginase, at a concentration of 100 to 5000 IU per milliliter of the composition; The concentration of disodium hydrogen phosphate in the composition is 0.1-0.8 wt.% by weight. Sodium dihydrogen phosphate has a concentration of 0.01-0.5 wt.% based on the weight of the composition; The concentration of the salt, based on the weight of the composition, is 0.1 to 1 wt.%. The sugar concentration, based on the weight of the composition, is from 0.5 to 20 wt.%. 5. The composition according to claim 1, comprising: Polyalkylene oxide-asparaginase, at a concentration of 100 to 5000 IU per milliliter of the composition; The composition contains disodium hydrogen phosphate at a concentration of 0.1-0.5 wt.% based on its weight. Sodium dihydrogen phosphate has a concentration of 0.01-0.1 wt.% based on the weight of the composition; The salt concentration, based on the weight of the composition, is 0.2 to 0.5 wt.%. The sugar concentration based on the weight of the composition is 1 to 10 wt.%. 6. The composition according to any one of claims 1-5, comprising: Polyalkylene oxide-asparaginase, at a concentration of 100 to 5000 IU per milliliter of the composition; The concentration of disodium hydrogen phosphate in the composition is 0.2-0.3 wt.% by weight. Sodium dihydrogen phosphate has a concentration of 0.05-0.07 wt.% based on the weight of the composition; The salt concentration, based on the weight of the composition, is 0.3 to 0.5 wt.%. The composition contains 4 to 5 wt.% sugar based on its weight. 7. A reconstructed lyophilized composition comprising: Polyalkylene oxide-asparaginase, at a concentration of 100 to 5000 IU per milliliter of the composition; The composition contains disodium hydrogen phosphate at a concentration of 0.2-0.3 wt.% based on its weight. Sodium dihydrogen phosphate has a concentration of 0.05-0.07 wt.% based on the weight of the composition; Sodium chloride, with a concentration of 0.3 to 0.5 wt.% based on the weight of the composition; and The composition contains 4 to 5 wt.% sucrose by weight. The polyalkylene oxide-asparaginase contains asparaginase covalently linked to a polyalkylene oxide group, and the polyalkylene oxide group contains a polyethylene glycol group. 8. The composition according to any one of claims 1-5, wherein the concentration of polyalkylene oxide-asparaginase is 500 to 2500 IU per milliliter of the composition, 500 to 1000 IU per milliliter of the composition, or 700 to 800 IU per milliliter of the composition. 9. The composition according to any one of claims 1-5, wherein the concentration of polyalkylene oxide-asparaginase is 750 μL per milliliter of the composition. 10. The composition of claim 9, wherein the polyethylene glycol group has a molecular weight of 2,000 to 10,000 Daltons. 11. The composition of claim 10, wherein the polyethylene glycol groups have a molecular weight of 5000 Daltons. 12. The composition of claim 1, wherein the polyalkylene oxide is covalently linked to asparaginase via a urethane linker. 13. The composition of claim 1, wherein the polyalkylene oxide is covalently linked to asparaginase via a succinate linker. 14. The composition according to any one of claims 1-5, wherein the composition further comprises sodium hydroxide and hydrochloric acid. 15. A freeze-dried, storage-stable composition comprising: Polyalkylene oxide-asparaginase; Buffer; Salt; and sugar, The freeze-dried, storage-stable composition is dehydrated and can be reconstituted with water to form the composition of any one of claims 1-14. 16. The freeze-dried, storage-stable composition of claim 15, wherein the reconstitution comprises combining the freeze-dried, storage-stable composition with water for injection (WFI). 17. A method for preparing the lyophilized polyalkylene oxide-asparaginase composition of claim 15 or 16 in a manner sufficient to produce a lyophilized, storage-stable polyalkylene oxide-asparaginase composition. 18. The method of claim 17, wherein the method comprises introducing an aqueous concentrate composition into a unit dose container and lyophilizing the aqueous concentrate composition in the unit dose container. 19. The method of claim 18, wherein the unit dose container is a vial. 20. The method of claim 19, wherein the method further comprises sealing the lyophilized composition in the unit dose container. 21. Use of the composition according to any one of claims 1-16 as a reconstituted dosage unit for the preparation of a medicament that deaminates asparagine in a subject. 22. The use according to claim 21, wherein the reconstitution comprises combining the lyophilized, storage-stable composition with water for injection (WFI). 23. The use according to claim 21, wherein the use is for treating the subject's tumor condition. 24. The use according to claim 23, wherein the tumor condition is cancer. 25. The use according to claim 24, wherein the cancer is leukemia. 26. The use according to claim 25, wherein the leukemia is acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). 27. The use according to any one of claims 21-23, wherein the subject has been prescribed a treatment regimen comprising an induction period, a consolidation period, and a maintenance period. 28. A medicine box comprising one or more unit dose containers, each container containing a lyophilized, storage-stable composition according to claim 15 or 16.