WO2020089743A1 - Pharmaceutical composition of pegylated l-asparaginase - Google Patents

Pharmaceutical composition of pegylated l-asparaginase Download PDF

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Publication number
WO2020089743A1
WO2020089743A1 PCT/IB2019/059119 IB2019059119W WO2020089743A1 WO 2020089743 A1 WO2020089743 A1 WO 2020089743A1 IB 2019059119 W IB2019059119 W IB 2019059119W WO 2020089743 A1 WO2020089743 A1 WO 2020089743A1
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WO
WIPO (PCT)
Prior art keywords
asparaginase
buffer
pegylated
pharmaceutical formulation
formulation
Prior art date
Application number
PCT/IB2019/059119
Other languages
French (fr)
Inventor
Sanjeev Kumar Mendiratta
Sanjay Bandyopadhyay
Chintan Patel
Tushar Patel
Chandresh Bhatt
Original Assignee
Cadila Healthcare Limited
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Publication date
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Publication of WO2020089743A1 publication Critical patent/WO2020089743A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)

Definitions

  • the present invention relates to stable pharmaceutical composition of L-asparaginase.
  • the composition is a stable lyophilized composition of pegylated L-asparaginase, preferably, a stable lyophilized composition of pegylated L-asparaginase free from salt(s).
  • L-asparaginase is an amidohydrolase which catalyzes L-asparagine into L-aspartic acid and ammonia.
  • L-asparaginase, pegylated L-asparaginase and their mechanism of metabolizing L- asparagine along with therapeutic role are described in WO2018017190.
  • Method of preparing pegylated L-asparaginase is well known in the art from patent document W01990013540.
  • Therapeutic proteins or polypeptides pose a number of challenges for pharmaceutical scientists regarding their formulation and delivery.
  • Maintaining the physical and chemical stability of protein or polypeptide molecules in solution is important to retain the biologically active conformation of the molecule, which results in providing the desired level of potency and safety of the pharmaceutical preparation for injection comprising the protein or polypeptide molecules.
  • Lack of physical and chemical stability may lead to significant degradation or irreversible modifications of protein or polypeptide molecules during processing, manufacturing, transportation and storage.
  • Protein aggregation or fragmentation or depegylation in pharmaceutical preparation is associated with loss of efficacy, altered pharmacokinetics, reduced stability, limited product shelf-life, and induction of unwanted immunogenicity.
  • a number of excipients have been used with varying success to reduce such protein degradation or modification.
  • each excipient has its own limitations, and in some cases, the more effective ones are observed to be less amenable to prepare a stable final formulation. Therefore, it is always challenging to establish stable formulation of sensitive protein or polypeptide molecules with a mixture of suitable inactive ingredients or excipients of interest, for pharmaceutical use.
  • a pegylated L-asparaginase liquid injection formulation (Oncaspar®) has been previously approved for commercial marketing by the U.S. Food and Drug Administration.
  • Oncaspar® was approved as a first-line treatment of patients with acute lymphoblastic leukemia (ALL) as a component of a multi-agent chemotherapy regimen.
  • ALL acute lymphoblastic leukemia
  • Oncaspar® was approved for the treatment of patients with ALL and hypersensitivity to asparaginase (e.g., native forms of L- asparaginase).
  • the approved liquid injection formulation of pegylated L-asparaginase comprises phosphate buffer and sodium chloride.
  • W02018017190 discloses a lyophilized formulation of pegylated L-asparaginase, comprising a therapeutically effective amount of pegylated L-asparaginase, buffer, salt and sugar.
  • the principal object of the present invention is to provide a stable pharmaceutical composition of L-asparaginase preferably, pegylated L-asparaginase comprising minimum possible excipients without compromising its biological activity.
  • the formulation according to the present invention is free from salt(s).
  • the formulation according to the present invention comprises a therapeutically effective amount of L-asparaginase preferably, pegylated L- asparaginase and one or more of sugar, buffer, amino acid optionally with other suitable excipient(s).
  • Such a formulation provides stability to the said molecule for therapeutic use either in single-dose or multi-dose form.
  • the present invention relates to stable pharmaceutical composition of pegylated L-asparaginase, comprising a therapeutically effective amount of pegylated L-asparaginase, sugar, buffer and/or amino acid optionally with other excipients for therapeutic use.
  • the current invention provides a lyophilized formulation of pegylated L-asparaginase free from salt(s).
  • Other aspects of the invention further include methods of making the composition.
  • the pharmaceutical formulation according to the present invention provides a stable composition of pegylated L- asparaginase, maintaining the structural integrity of protein even after multiple freeze-thaw cycles.
  • the composition according to the present invention is stable which can withstand at least two freeze-thaw cycle, more preferably, five freeze-thaw cycle.
  • Figure 1 illustrates purity of pegylated L-asparaginase (Example 1) by analytical HP-size exclusion chromatography (HP-SEC).
  • Figure 2 illustrates purity of pegylated L-asparaginase (Example 2) by analytical HP-size exclusion chromatography (HP-SEC).
  • Figure 3 illustrates purity of pegylated L-asparaginase (Example 3) by analytical HP-size exclusion chromatography (HP-SEC).
  • Figure 4 illustrates purity of pegylated L-asparaginase (Example 4) by analytical HP-size exclusion chromatography (HP-SEC).
  • Figure 5 illustrates purity of pegylated L-asparaginase (Example 5) by analytical HP-size exclusion chromatography (HP-SEC).
  • Figure 6 illustrates purity of pegylated L-asparaginase (Example 6) by analytical HP-size exclusion chromatography (HP-SEC).
  • HP-SEC High pressure size exclusion chromatography
  • RI detector Refractive index detector
  • L-asparaginase represents an amidohydrolase which catalyzes L-asparagine into L- aspartic acid and ammonia. It also includes pegylated form of L-asparaginase (pegylated L- asparaginase). Said pegylated form of L-asparaginase includes L-asparaginase which is covalently conjugated to monomethoxypolyethylene glycol (mPEG).
  • mPEG monomethoxypolyethylene glycol
  • pegylated L- asparaginase is pegaspargase and calaspargase sold under brand name Oncaspar ® and AsparlasTM respectively.
  • composition e.g., an aqueous formulation or a lyophilized formulation that it is useful for treating a disease or disorder.
  • therapeutic amount refers to the amount of therapeutic agent used to treat disorder or disease without causing toxicity to the patients.
  • the therapeutic agent according to the present invention is pegylated L-asparaginase or L-asparaginase.
  • pharmaceutical formulation refers to preparations, which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective.
  • pharmaceutical formulation or“pharmaceutical composition” or“composition” can be used here interchangeably.
  • The“lyophilized formulation” is a dosage form, which is prepared by lyophilization or freeze drying process.
  • the lyophilization was performed with lyophilization technique involving steps such as freezing, primary drying, secondary drying and optionally annealing.
  • freezing is rapid freezing as described herein.
  • The“frozen composition of L-asparaginase or pegylated L-asparaginase” is a dosage form in which freezing of aqueous solution of L-asparaginase or pegylated L-asparaginase is conducted at or below -15 °C.
  • excipient refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain osmolality and pH of the pharmaceutical preparations.
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.
  • “Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
  • salt according to the present invention is a component which is not a part of a buffer system. Preferably, such salts are inorganic salts which is not a part of buffer system.
  • inorganic salt refers to any salt that does not contain C-H bonds as opposed to organic salt that contains C-H bonds.
  • Inorganic salts such as sodium chloride, potassium chloride etc. are widely used as stabilizers or tonicity agents in addition to their use in a buffer system in the pharmaceutical formulations.
  • examples of inorganic salts include, but are not limited to, sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCl 2 ), barium chloride (BaCE), ferric chloride (FeCE) etc.
  • the term“free from salt” refers to the composition of pegylated L- asparaginase which does not include salt in addition to buffer.
  • the salt referred in“free from salt” is the salt that has no buffering capacity. According to the present invention, preferably, inorganic salt is not added as an excipient.
  • “buffer” or“buffer solution” or“buffer system” are well known to a person skilled in the art and generally refers to an aqueous solution comprising a mixture of an acid (usually a weak acid, e.g. succinic acid) and its conjugate base (e.g. succinate or sodium succinate) or alternatively a mixture of a base (usually a weak base, e.g. histidine) and its conjugate acid (e.g. protonated histidine salt).
  • a buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the“buffering agent(s)”.
  • the pH of a "buffer solution” will change very only slightly upon addition of a small quantity of strong acid or base due to the “buffering effect” imparted by the“buffering agent(s)”.
  • buffer system is well known to a person skilled in the art and generally comprises one or more buffering agent(s) and/or an acid/base conjugate(s) thereof, and more suitably comprises one buffering agent only and an acid/base conjugate thereof.
  • any concentrations stipulated herein in relation to a“buffer system” i.e. a buffer concentration
  • concentrations stipulated herein in relation to a“buffer system” suitably refer to the combined concentration of all the relevant buffering species (i.e. the species in dynamic equilibrium with one another, e.g. succinate / succinic acid).
  • a given concentration of a succinate buffer system generally relates to the combined concentration of succinate (or succinate salt(s), e.g. sodium succinate) and its corresponding acid. Skilled person can also use other suitable acid for the preparation of buffer system.
  • the overall pH of the composition comprising the relevant buffer system is generally a reflection of the equilibrium concentration of each of the relevant buffering species (i.e. the balance of buffering agent(s) to acid / base conjugate(s) thereof).
  • buffering agent is well known to a person skilled in the art and generally refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution.
  • a buffering agent maintain the pH of a given solution at or near to a pre-determined value, and the buffering agents are generally chosen to complement the pre-determined value.
  • a buffering agent is suitably a single compound which gives rise to a desired buffering effect, especially when said buffering agent is mixed with (and suitably capable of proton exchange with) an appropriate amount (depending on the pre-determined pH desired) of its corresponding “acid/base conjugate”, or if the required amount of its corresponding“acid / base conjugate” is formed in- situ which can be achieved by adding strong acid or base until it attains the required pH.
  • the term“acid / base conjugate” is well known to a person skilled in the art and generally refers to the conjugate acid or conjugate base (whichever is relevant at a particular pH - typically the conjugate acid in the context of the present invention) of a particular "buffering agent".
  • the acid / base conjugate of a succinate buffering agent e.g. sodium succinate
  • succinic acid is suitably succinic acid.
  • buffering species refers to the particular species (excluding any associated counteranions or countercations - i.e. ignore sodium ions for sodium succinate / succinic acid systems) of a given buffer system which are in dynamic equilibrium with (and proton-exchange with) one another.
  • succinate anions and succinic acid together constitute the “succinate buffering species” of a“succinate buffer system”.
  • high molecular weight species or“low molecular weight species” are product related substances that contributes to the size heterogeneity of a protein.
  • the present invention protein is pegylated L-asparaginase.
  • High molecular weight species may include dimers, trimers, or aggregates of a desired protein, pegylated and / or non-pegylated form of desired protein, preferably pegylated L-asparaginase, as well as undesired polypeptide contaminants.
  • LMW may include free unmodified asparaginase, depegylated or fragmented asparaginase.
  • A“disorder” or“disease” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions, which predisposes the subject to the disorder in question.
  • a“therapeutically effective amount” or“effective amount” of an antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the antibody is effective.
  • water is intended to mean water that has been purified to remove contaminants, usually by distillation or reverse osmosis, also referred to herein as“pure water”.
  • water used in the methods and compositions of the invention is excipient-free.
  • water includes sterile water suitable for administration to a subject.
  • water is meant to include water for injection (WFI).
  • patient and“subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both humans and non-human animals.
  • 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 gallinaceous birds, ducks, geese, and the like.
  • the term does not denote a particular age. Thus, adult, juvenile and newborn individuals are of interest.
  • the invention provides pharmaceutical formulation comprising a therapeutic amount of L-asparaginase.
  • the invention provides pharmaceutical formulation comprising a therapeutic amount of pegylated L-asparaginase.
  • the pharmaceutical formulation of L-asparaginase according to the present invention is a liquid formulation or lyophilized formulation.
  • the pharmaceutical formulation of L-asparaginase according to the present invention is a lyophilized formulation.
  • the pharmaceutical formulation of pegylated L-asparaginase according to the present invention is a liquid formulation or lyophilized formulation.
  • the pharmaceutical formulation of pegylated L-asparaginase according to the present invention is a lyophilized formulation.
  • the lyophilized formulation according to the present invention comprises L- asparaginase with one or more suitable excipient(s).
  • the lyophilized formulation according to the present invention comprises pegylated L-asparaginase with one or more suitable excipient(s).
  • the present invention provides a pharmaceutical formulation suitable for human use which comprises a therapeutic amount of L-asparaginase, sugar and other suitable excipient(s) optionally selected from buffer, amino acid, surfactant, anti-oxidant, anti-microbial agent and WFI.
  • the present invention provides a pharmaceutical formulation suitable for human use which comprises L-asparaginase and a suitable sugar.
  • the present invention provides pharmaceutical formulation of L- asparaginase comprises buffer.
  • the pharmaceutical formulation of L-asparaginase according to the present invention comprises one or more amino acid.
  • the present invention provides pharmaceutical formulation of L- asparaginase comprises suitable sugar, buffer and optionally one or more amino acid and/or surfactant.
  • the present invention provides a pharmaceutical formulation suitable for human use which comprises a therapeutic amount of pegylated L-asparaginase, sugar and other suitable excipient(s) optionally selected from buffer, amino acid, surfactant, anti-oxidant, anti- microbial agent and WFI.
  • the present invention provides a pharmaceutical formulation suitable for human use which comprises pegylated L-asparaginase and suitable sugar.
  • the present invention provides pharmaceutical formulation of pegylated L-asparaginase comprises buffer.
  • the pharmaceutical formulation of pegylated L-asparaginase according to the present invention comprises one or more amino acid.
  • the present invention provides pharmaceutical formulation of pegylated L-asparaginase comprising suitable sugar, buffer and optionally one or more amino acid and/or surfactant.
  • the method of lyophilizing an aqueous composition that comprises L- sparaginase, a buffer, a sugar optionally an amino acid and/or surfactant in a manner sufficient to produce stable lyophilized L-asparaginase composition.
  • the method of lyophilizing an aqueous composition that comprises a pegylated L-asparaginase, a buffer, a sugar optionally an amino acid and / or surfactant in a manner sufficient to produce stable lyophilized pegylated L-asparaginase composition.
  • the lyophilized formulation according to the present invention is reconstituted with water.
  • the pharmaceutical formulation of L-asparaginase is free from salt(s). In a preferred embodiment, the pharmaceutical formulation of L-asparaginase is free from inorganic salt(s). In one of the embodiments, the pharmaceutical formulation of pegylated L-asparaginase is free from salt(s). In a preferred embodiment, the pharmaceutical formulation of pegylated L- asparaginase is free from inorganic salt(s). In a preferred embodiment, the pharmaceutical formulation of L-asparaginase comprising a therapeutic amount of L-asparaginase, sugar and buffer optionally with one or more suitable excipients, wherein the formulation is free from salt.
  • the pharmaceutical formulation of pegylated L-asparaginase comprising a therapeutic amount of pegylated L-asparaginase, sugar and buffer optionally with one or more suitable excipients, wherein the formulation is free from salt.
  • the aqueous formulation of present invention comprises L-asparaginase in water. In one more embodiment, the aqueous formulation of present invention comprises pegylated L- asparaginase in water.
  • the aqueous pharmaceutical formulation according to the present invention further comprises one or more excipients selected from a sugar, buffer, amino acid, surfactant and the like.
  • the pharmaceutical formulation of present invention maintains structural integrity of L-asparaginase upon multiple freezing cycles.
  • the pharmaceutical formulation of present invention maintains structural integrity of L- asparaginase at least after two freeze-thaw cycle, more preferably, at least after five freeze-thaw cycle.
  • a pharmaceutical formulation of L-asparaginase according to the present invention wherein L-asparaginase is pegylated L-asparaginase or pegaspargase or calaspargase.
  • the pharmaceutical formulation of present invention maintains structural integrity of pegylated L-asparaginase upon multiple freeze-thaw cycles. In a preferred embodiment, the pharmaceutical formulation of present invention maintains structural integrity of pegylated L-asparaginase at least after two freeze-thaw cycle, more preferably, at least after five freeze-thaw cycle. In one of the embodiments, the pharmaceutical formulation of present invention contains low level of bacterial endotoxins. In a preferred embodiment, the pharmaceutical formulation of present invention contains less than 100 EU / mg bacterial endotoxins.
  • the method of preparing lyophilized formulation of the present invention comprises the steps of freezing, primary drying, secondary drying and optionally with the step of annealing.
  • the present invention provides method of preparing lyophilized formulation of L-asparaginase or pegylated L-asparaginase comprising steps of freezing, primary drying and secondary drying optionally annealing, wherein freezing is performed at a relatively faster rate to achieve the desired freezing temperature.
  • method of preparing lyophilized formulation of pegylated L- asparaginase of the present invention comprises following steps:
  • the present invention provides a stable composition of L-asparaginase, preferably a stable composition of pegylated L-asparaginase.
  • the composition according to the present invention includes one or more of a sugar, buffer and an amino acid as an excipient along with L- asparaginase.
  • the current invention further provides methods of making the composition of L- asparaginase, preferably of pegylated L-asaparaginase.
  • the composition according to the present invention finds therapeutic use in single-dose form or in multi-dose form.
  • the present invention includes stable frozen or lyophilized L- asparaginase composition, preferably pegylated L- asparaginase composition.
  • the frozen or lyophilized composition of L-asparaginase or pegylated L-asaparaginase according to the present invention comprises one or more of a sugar, buffer and an amino acid.
  • the lyophilization process to prepare formulation of the present invention is performed using the techniques available in the art, which includes various steps such as freezing preferably rapid freezing, primary drying, secondary drying and optionally annealing.
  • the formulation of the invention has improved stability either in frozen or lyophilized or liquid form.
  • the formulation of the invention maintains stability after at least one freeze- thaw cycle.
  • composition e.g., frozen or lyophilized or liquid
  • methods of making composition of present invention are described in details.
  • the present invention provides stable pharmaceutical composition comprising therapeutic amount of L-asparaginase, preferably therapeutic amount of pegylated L-asparaginase.
  • the pegylated L-asparaginase according to the present invention is an L-asparaginase covalently linked to polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the pegylated L-asparaginase may include PEG covalently linked to the asparaginase at one or more positions on the asparaginase.
  • PEG may be covalently linked to an amino acid residue of the asparaginase.
  • the PEG is covalently linked to an amine group of an amino acid residue of the asparaginase.
  • the PEG is covalently linked to an amino acid side chain of an N-terminal amino acid in the asparaginase. In some embodiments, the PEG is covalently linked to an epsilon-amino group of lysine in the asparaginase. In some embodiments, the PEG is covalently linked to an amino acid side chain of the N-terminal amino acid and an epsilon-amine group of lysine(s) in the asparaginase. In some instances, the pegylated L-asparaginase is substantially non-antigenic. In some instances, the pegylated L-asparaginase has a reduced rate of clearance from the systemic circulation of a subject as compared to an unmodified L-asparaginase.
  • the pharmaceutical formulation of L-asparaginase or pegylated L-asparaginase according to present invention may be administered to the subject in particular dosage.
  • the “dosage” according to the present invention refers to amount of active ingredient administered to the subject in single dose or multiple dose.
  • the dosage unit includes an amount of pegylated L-asparaginase ranging from 500 to 1,000 IU / mL.
  • the dosage unit includes an amount of the pegylated L-asparaginase ranging from 700 to 800 IU / mL.
  • the amount of pegylated L-asparaginase in composition according to the present invention is about 750 IU / mL.
  • compositions of the present disclosure in addition to the pegylated L- asparaginase, may include carbohydrate.
  • the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising carbohydrate.
  • a carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may also be employed.
  • carbohydrate excipients of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.
  • monosaccharides such as fructose, maltose, galactose, glucose, mannose, sorbose, and the like
  • disaccharides such as lac
  • the composition may include a sugar.
  • Preferred sugars suitable for use in the compositions of the present invention include sugars that are compatible with the L- asparaginase as well as compatible with pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration.
  • suitable sugars include, but not limited to, sucrose, mannitol, sorbitol, maltose, trehalose, hydroxypropyl-beta- cyclodextrin (b-HPCD), lactose, glucose, fructose, galactose, glucosamine, and the like, and combinations thereof.
  • the sugar is a disaccharide.
  • the disaccharide may be sucrose or trehalose.
  • the sugar is a sugar alcohol.
  • the sugar alcohol may be sorbitol or mannitol.
  • the suitable sugar comprises trehalose, sucrose or mannitol or suitable combinations thereof.
  • the present invention provides a composition of F-asparaginase or pegylated F-asparaginase comprising sugar selected from monosaccharides, disaccharides, polysaccharides and alditols.
  • the present invention provides a composition of F-asparaginase or pegylated F-asparaginase comprising sugar selected from fructose, maltose, galactose, glucose, mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrins, dextrans, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myoinositol and suitable combinations thereof.
  • sugar selected from fructose, maltose, galactose, glucose, mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrins, dextrans, mannitol, xylit
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar selected from lactose, sucrose, trehalose, sorbitol, mannitol, raffinose, and suitable combinations thereof.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar selected from sucrose, trehalose, mannitol and suitable combinations thereof.
  • the sugar concentration according to the present invention is in the range of about 0.05 mg / mL to 150 mg / mL.
  • the sugar concentration according to the present invention is in the range of about 0.1 mg / mL to 100 mg / mL.
  • the sugar concentration according to the present invention is in the range of about 0.05 mg / mL to 20 mg / mL or about 0.05 mg / mL to 50 mg / mL or about 50 mg / mL to 75 mg / mL or about 50 mg / mL to 100 mg / mL or about 40 mg / mL to 90 mg / mL or 50 mg / mL to 150 mg / mL.
  • the concentration range according to present disclosure include each integer and non-integer number between particular ranges.
  • the concentration range of sugar about 0.05 mg / mL to 150 mg / mL include 0.05 mg / mL, 0.06 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 5.0 mg / mL, 10.5 mg / mL, 50.0 mg / mL, 60.0 mg / mL, 75.0 mg / mL, 85.0 mg / mL, 100.0 mg / mL, 125.0 mg / mL, 150.0 mg / mL or any integer or non-integer number in between them.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in the range of 40 mg / mL to 90 mg / mL. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in the range of 60 mg / mL to 90 mg / mL.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in amount of 60 mg / mL or 65 mg / mL or 75 mg / mL or 85 mg / mL or 90 mg / mL.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sucrose at a concentration of about 65 mg / mL or about 75 mg / mL or about 85 mg / mL or about 90 mg / mL.
  • the present invention provides a composition of L- asparaginase or pegylated L-asparaginase comprising trehalose at a concentration of about 60 mg / mL.
  • Another excipient that may be included in the compositions of the present disclosure is a buffer.
  • Buffers suitable for use in the compositions of the present disclosure include buffers that are compatible with the L-asparaginase as well as with the pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration.
  • suitable buffers include, but are not limited to, phosphate buffer, histidine buffer, glycine buffer, arginine buffer, citrate buffer, succinate buffer, acetate buffer, tromethamine buffer, bicarbonate buffer, carbonate buffer, sulfate buffer, chloride buffer and suitable combinations thereof and the like.
  • the suitable buffer according to the present is selected from phosphate buffer, histidine buffer, glycine buffer, arginine buffer, citrate buffer, succinate buffer, acetate buffer, tromethamine buffer, bicarbonate buffer, carbonate buffer, sulfate buffer, chloride buffer and suitable combinations thereof.
  • the suitable buffer according to the present invention is selected from phosphate buffer, histidine buffer, succinate buffer and suitable combination thereof.
  • the suitable buffer according to the present invention is a phosphate buffer or a succinate buffer.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer wherein buffer is succinate buffer or phosphate buffer.
  • the buffer included in the composition according to the present invention may be a buffer that maintains the pH of the composition at a pH, ranging from about pH 5.0 to about pH 8.0, or a pH of about 7.0.
  • the pH range 5.0 to 8.0 according to the present invention includes pH 5.0, pH 6.0, pH 7.0 or pH 8.0 or any non-integer number in between them.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH in the range of pH 5.0 to pH 8.0.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH in the range of pH 6.0 to pH 7.5.
  • pH 6.0 to pH 7.5 according to the present invention includes pH 6.0, pH 6.5, pH 7.0, pH 7.3, pH 7.5 or any non-integer or integer number in between them.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH 7.3 or pH 6.5.
  • the buffer concentration according to the present invention is in the range of about 5 mM to 100 mM. In a preferred embodiment, the buffer concentration is in the range of about 5 mM to 75 mM.
  • the concentration range of buffer according to present disclosure include each integer and non-integer number between particular range. Lor example, the concentration range of about 5 mM to 100 mM include 5.0 mM or 10.0 mM or 20.0 mM or 30.0 mM or 40.0 mM or 50.0 mM or 60.0 mM or 70.0 mM or 80 mM or 90 mM or 100 mM or any integer or non integer number in between them.
  • the concentration range of buffer in millimolar (mM) as mentioned above according to the present invention includes corresponding equivalent concentration in mg / mL.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer in the range of about 5 mM to about 49 mM.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer at a concentration, 10 mM or 25 mM or 49 mM.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising phosphate buffer at a concentration, 25 mM or 49 mM. In one more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising succinate buffer at a concentration, 10 mM.
  • amino acid Another excipient that may be included in the compositions of the present disclosure is an amino acid.
  • Amino acids suitable for use in the compositions of the present disclosure include amino acids that are compatible with the L-asparaginase as well as with the pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration.
  • suitable amino acids include, but are not limited to, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine / cystine and the like, their suitable salt form or suitable combinations of any of the above.
  • the suitable amino acid is selected from arginine, glycine, leucine, lysine, histidine, methionine, glutamic acid or their suitable salt form or combinations, thereof.
  • Amino acid according to the present invention can be a part of buffer as a buffering agent or buffering species or acid / base conjugate.
  • the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising amino acid selected from arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine / cysteine, their suitable salt form and combination thereof.
  • the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising amino acid selected from arginine, glycine, leucine, lysine, histidine, methionine, glutamic acid, their suitable salt form and suitable combinations thereof.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising L-arginine or glycine or suitable combinations thereof.
  • the amino acid according to the present invention is present in amount about 0.1 mg / mL to about 100 mg / mL, preferably the amino acid is present in amount about 0.1 mg / mL to about 50 mg / mL, more preferably the amino acid is present in amount about 0.1 mg / mL to about 10 mg / mL.
  • concentration range according to present disclosure include each integer and non-integer number between a particular range.
  • the concentration range of about 0.1 mg / mL to about 100 mg / mL include 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 5.0 mg / mL, 10.5 mg / mL, 50.0 mg / mL, 75.0 mg / mL, 100.0 mg / mL or any integer or non-integer number in between them.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising amino acid in the range of 0.1 mg / mL to 100 mg / mL, preferably 0.5 mg / mL to 10.0 mg / mL.
  • the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising L- arginine at a concentration 5 mg / mL. In a one more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising glycine at a concentration 5 mg / mL.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising a sugar, a buffer and optionally an amino acid, in addition to the pegylated L-asparaginase.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar and buffer in addition to the pegylated L-asparaginase.
  • the present invention provides a lyophilized composition of L-asparaginase or pegylated L-asparaginase comprising sugar and buffer in addition to the pegylated L-asparaginase.
  • preferred aspects of the present disclosure include a lyophilized storage stable composition of a pegylated L- asparaginase comprises suitable amount of trehalose or sucrose or raffinose or sorbitol or lactose or mannitol as a sugar, succinate or phosphate or histidine as a buffer and optionally arginine or glycine or methionine or histidine or glutamic acid as an amino acid.
  • the present invention provides a stable lyophilized composition of L- asparaginase or pegylated L-asparaginase comprising pegylated L- asparaginase, sugar and buffer.
  • the present invention provides a stable lyophilized composition comprising pegylated L- asparaginase, sucrose or trehalose, phosphate buffer or succinate buffer optionally with one or more excipients as described in the specification.
  • compositions of the present disclosure may also include other components, such as additional pharmaceutically acceptable excipients or a dosage delivery vehicle as part of the composition.
  • Additional excipients according to the present invention may include, but are not limited to, antimicrobial agents, anti-oxidants, surfactants, water (e.g., water for injection (WFI)), alcohols, glycerin, vegetable oils, phospholipids and any combinations thereof.
  • WFI water for injection
  • surfactants include, but not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene -polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), sodium dodecyl sulphate (SDS) and the like.
  • the suitable surfactant is polyoxyethylensorbitan-fatty acid esters (Tweens).
  • the polyoxyethylensorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM), polysorbate 80 (sold under the trademark Tween 80TM) and polyethylene -polypropylene copolymers which are sold under the names Pluronic (R) F68 or Poloxamer 188TM.
  • the present invention provides a composition of L- asparaginase or pegylated L-asparaginase free from salt.
  • the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising L-asparaginase or pegylated L-asparaginase as an active ingredient, sugar, buffer and wherein the composition is free from salt.
  • the salt according to the present invention which is not present may be selected from sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCL), barium chloride (BaCL), ferric chloride (FeCF).
  • composition of pegylated L- asparaginase known in the art includes salt in addition to buffer and sugar while the composition of pegylated L-asparaginase according to the current invention does not include salt in addition to buffer. According to the present invention, salts, that have no buffering capacity, are not added in composition of pegylated L-asparaginase.
  • the composition according to the present invention is stable and can provide long-term stability. It is illustrated herein below examples by providing analytical data of stability analysis at various temperature and at various forms of composition by HP-SEC.
  • the composition (e.g., frozen or lyophilized or liquid) is a sterile composition.
  • sterile is meant that there are substantially no microbial contaminants (e.g., fungi, bacteria, viruses, spore forms, etc.).
  • the composition e.g., frozen or lyophilized or liquid
  • the present invention provides pharmaceutical composition of pegylated L-asparaginase that is substantially free of particles and/or aggregates and prevents the formation of particles and/or aggregates during storage and/or transport.
  • the present invention provides pharmaceutical composition of pegylated L-asparaginase that is stable for at least one month at storage condition between +2 °C and +8 °C, at least one month at storage condition of 30 °C ⁇ 2 °C and RH 65 % ⁇ 5 and at least one month at storage condition of 40 °C ⁇ 2 °C and RH 75 % ⁇ 5. It is illustrated herein below examples by providing analytical data of stability analysis at various temperature and at various forms of composition by HP-SEC. Such stable formulation is prepared with specific lyophilization process as described in this application.
  • Method of preparing lyophilized formulation comprising freezing preferably rapid freezing, annealing, primary drying and secondary drying is performed preferably in 40 - 120 hours.
  • Pegylated L-asparaginase solution filled in USP Type I glass vials is lyophilized after loading the vials in a lyophilizer, from ambient temperature to +5 °C, followed by freezing at around - 50 °C which is achieved in less than an hour, at a relatively faster rate than the process known in the art for lyophilization of L-asparaginase or pegylated L-asparaginase.
  • the frozen mass in the vial is held for another at least 1 hour at the same temperature and undergoes primary drying cycle, under vacuum at 40 - 60 mTorr for at least 24 hours at a temperature range from about - 50 °C to - 20 °C.
  • Lollowing primary drying, secondary drying cycle is carried out at relatively higher temperature between +20 °C and +30 °C for at least about 24 hours.
  • vials are fully stoppered under No and unloaded at ambient pressure and temperature.
  • Samples were analyzed to determine the purity, level of HMW and LMW species in pegylated L- asparaginase preparations by HP-SEC using YMC Pak Diol-300 column (8.0 mm I.D x 30 cm L). Samples containing pegylated L- asparaginase were loaded and eluted isocratic in mode using sodium phosphate buffer containing 2-propranol (90: 10 v/v) at a flow rate of 0.5 mL / min. Elution was monitored at UV 214 nm for the protein peaks. Free PEG was determined with RI detection.
  • Samples were analyzed by denaturing gel electrophoresis under non-reducing conditions on 4 - 20 % polyacrylamide gel and detection of pegylated protein performed by using coomassie brilliant blue solution R250 staining solution.
  • L-asparaginase activity in pegylated form was determined by estimating the ability of the enzyme to effectively convert the substrate, L-asparagine, to aspartate and ammonia.
  • the quantitative determination of the released ammonia directly correlates with the effective amount of active enzyme present in pegylated L-asparaginase.
  • the UV absorbance of pegylated L-asparaginase samples were measured at 280nm in 1 cm path length on UV spectrophotometer to check the concertation of pegylated L-asparaginase.
  • the respective formulation buffer was used as blank.
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration(s), as described above. pH of the formulation medium was maintained at around pH 7.30. Excipient(s) were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with sterile water or Water for Injection. The formulated pegylated L-asparaginase was evaluated for stability upon repeated freezing and thawing as shown in Table 13. Formulated samples were analyzed for purity by HP- SEC. Purity of pegylated L-asparaginase sample after formulation was found to be 99.81 % as shown in Figure 1.
  • a person skill in the art can also formulate the composition for pegylated L- asparaginase and can fill in the suitable container-closure system (like vials, cartridges, syringes etc.) for storage. Lyophilization of the formulated sample was carried out in the above specified container-closure systems. Lyophilization was performed in about 110 hours. Pegylated L- asparaginase solution filled in USP Type I glass vials was lyophilized after loading the vials in a lyophilizer, from ambient temperature to +5 °C, followed by freezing at around - 45 °C which is achieved in less than an hour.
  • a person skilled in the art can further evaluate the stability of formulated pegylated L- asparaginase at various temperature conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed storage conditions (higher temperature; about +40 °C).
  • Real-Time storage condition between +2 °C and +8 °C
  • Accelerated storage condition about +25 °C
  • stressed storage conditions higher temperature; about +40 °C.
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose and L- arginine at desired concentration(s), as described above. pH of the formulation medium was maintained at around pH 7.30. Excipient(s) were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with sterile water or Water for Injection. The formulated pegylated L-asparaginase was evaluated for stability upon repeated freezing and thawing as shown in Table 13. Formulated samples were analyzed for purity by HP-SEC.
  • pegylated L-asparaginase sample after formulation was found to be 99.81 % as shown in Figure 2.
  • a person skill in the art can also formulate the composition for pegylated L-asparaginase and can fill in the suitable container-closure system (like vials, cartridges, syringes etc.) for storage. Lyophilization of the formulated sample was performed as described in example 1.
  • a person skilled in the art can further evaluate the stability of formulated pegylated L-asparaginase at various temperature conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed storage conditions (higher temperature; about +40 °C).
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 7.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed with as described in example 1. Table 1 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 3. Purity of pegylated L-asparaginase after formulation was found to be 99.13 %, as shown in Figure 3.
  • Stability of lyophilized pegylated L-asparaginase prepared with example 3 was evaluated over a period of 30 days (1 month), under various storage conditions, such as Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (30 °C ⁇ 2 °C; RH 65 % ⁇ 5 %), and Stressed temperature condition (40 °C ⁇ 2 °C; RH 75 % ⁇ 5 %). Lyophilized samples exposed to different temperature conditions were withdrawn at different time points, reconstituted and analyzed for purity by HP-SEC, as indicated in Table 2 (Real-Time), Table 3 (Accelerated) and Table 4 (Stressed). Table 5 illustrates the stability (in-use) of reconstituted pegylated L-asparaginase stored between +2 °C and +8 °C, as assessed by HP-SEC for purity.
  • Table 5 Stability of reconstituted pegylated L-asparaginase stored between +2 °C and +8
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in succinate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 6.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 6 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 4. Purity of pegylated L- asparaginase after formulation was found to be 99.12 %, as shown in Figure 4.
  • Stability of lyophilized pegylated L-asparaginase prepared with example 4 was evaluated over a period of 30 days (1 month), under various storage conditions, such as Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (30 °C ⁇ 2 °C; RH 65 % ⁇ 5 %), and Stressed temperature condition (40 °C ⁇ 2 °C; RH 75 % ⁇ 5 %). Lyophilized samples exposed to different temperature conditions were withdrawn at different time points, reconstituted and analyzed for purity by HP-SEC, as indicated in Table 7 (Real-Time), Table 8 (Accelerated) and Table 9 (Stressed). Table 10 illustrates the stability (in-use) of reconstituted pegylated L-asparaginase stored between +2 °C and +8 °C, as assessed by HP-SEC for purity.
  • Table 7 Purity of lyophilized pegylated L-asparaginase stored under Real-Time condition as assessed by HP-SEC - Example 4
  • Table 8 Purity of lyophilized pegylated L-asparaginase stored under Accelerated condition as assessed by HP-SEC - Example 4
  • Example 3 the lyophilized formulations, described with Example 3 and Example 4 were found to be stable under various storage conditions, as assessed by HP-SEC for purity of pegylated L- asparaginase.
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 7.30. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 11 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 4. Purity of pegylated L- asparaginase after formulation was found to be 99.22 %, as shown in Figure 5. Table 11: Results obtained for physicochemical properties and enzyme activity of pegylated
  • Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in succinate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 6.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 12 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 6. Purity of pegylated L- asparaginase after formulation was found to be 99.26 %, as shown in Figure 6.
  • Formulations as mentioned in Example 1, Example 2 and Examples 7 - 17, as prepared herein and shown above were exposed to repeated freeze-thaw cycles (5 cycles) to check stability of pegylated L-asparaginase upon freezing and thawing. Samples after repeated freezing and thawing were analyzed for purity by HP-SEC. Results obtained with pegylated L-asparaginase formulated samples after the I st and 5 th freeze-thaw cycles are shown in Table 14. None of the formulation composition was found to show any significant increase in HMW or LMW species. All samples were observed to show greater than 99 % purity even after the 5 th freeze-thaw cycle.

Abstract

Pharmaceutical composition of L-asparaginase The present invention relates to stable pharmaceutical composition of L-asparaginase. In some instances, the composition is a stable lyophilized composition of pegylated L-asparaginase. Preferably, the composition is a stable lyophilized composition of pegylated L-asparaginase free from salt. In some instances, the lyophilized composition includes one or more of a sugar, buffer and amino acid. Aspects of the invention further include method of making the compositions.

Description

“PHARMACEUTICAL COMPOSITION OF PEGYLATED L-ASPARAGINASE”
Field of the Invention
The present invention relates to stable pharmaceutical composition of L-asparaginase. In some instances, the composition is a stable lyophilized composition of pegylated L-asparaginase, preferably, a stable lyophilized composition of pegylated L-asparaginase free from salt(s).
Background of the invention
L-asparaginase is an amidohydrolase which catalyzes L-asparagine into L-aspartic acid and ammonia. L-asparaginase, pegylated L-asparaginase and their mechanism of metabolizing L- asparagine along with therapeutic role are described in WO2018017190. Method of preparing pegylated L-asparaginase is well known in the art from patent document W01990013540. Therapeutic proteins or polypeptides pose a number of challenges for pharmaceutical scientists regarding their formulation and delivery. Maintaining the physical and chemical stability of protein or polypeptide molecules in solution is important to retain the biologically active conformation of the molecule, which results in providing the desired level of potency and safety of the pharmaceutical preparation for injection comprising the protein or polypeptide molecules. Lack of physical and chemical stability may lead to significant degradation or irreversible modifications of protein or polypeptide molecules during processing, manufacturing, transportation and storage. Protein aggregation or fragmentation or depegylation in pharmaceutical preparation is associated with loss of efficacy, altered pharmacokinetics, reduced stability, limited product shelf-life, and induction of unwanted immunogenicity. In pharmaceutical preparation of protein drug product, a number of excipients have been used with varying success to reduce such protein degradation or modification. However, each excipient has its own limitations, and in some cases, the more effective ones are observed to be less amenable to prepare a stable final formulation. Therefore, it is always challenging to establish stable formulation of sensitive protein or polypeptide molecules with a mixture of suitable inactive ingredients or excipients of interest, for pharmaceutical use.
A pegylated L-asparaginase liquid injection formulation (Oncaspar®) has been previously approved for commercial marketing by the U.S. Food and Drug Administration. Oncaspar® was approved as a first-line treatment of patients with acute lymphoblastic leukemia (ALL) as a component of a multi-agent chemotherapy regimen. In addition, Oncaspar® was approved for the treatment of patients with ALL and hypersensitivity to asparaginase (e.g., native forms of L- asparaginase). The approved liquid injection formulation of pegylated L-asparaginase comprises phosphate buffer and sodium chloride.
W02018017190 discloses a lyophilized formulation of pegylated L-asparaginase, comprising a therapeutically effective amount of pegylated L-asparaginase, buffer, salt and sugar.
The principal object of the present invention is to provide a stable pharmaceutical composition of L-asparaginase preferably, pegylated L-asparaginase comprising minimum possible excipients without compromising its biological activity. Preferably, the formulation according to the present invention is free from salt(s). The formulation according to the present invention comprises a therapeutically effective amount of L-asparaginase preferably, pegylated L- asparaginase and one or more of sugar, buffer, amino acid optionally with other suitable excipient(s). Such a formulation provides stability to the said molecule for therapeutic use either in single-dose or multi-dose form.
Summary of the invention
The present invention relates to stable pharmaceutical composition of pegylated L-asparaginase, comprising a therapeutically effective amount of pegylated L-asparaginase, sugar, buffer and/or amino acid optionally with other excipients for therapeutic use. Preferably, the current invention provides a lyophilized formulation of pegylated L-asparaginase free from salt(s). Other aspects of the invention further include methods of making the composition. The pharmaceutical formulation according to the present invention provides a stable composition of pegylated L- asparaginase, maintaining the structural integrity of protein even after multiple freeze-thaw cycles. Preferably, the composition according to the present invention is stable which can withstand at least two freeze-thaw cycle, more preferably, five freeze-thaw cycle. Brief description of drawings
Figure 1 illustrates purity of pegylated L-asparaginase (Example 1) by analytical HP-size exclusion chromatography (HP-SEC).
Figure 2 illustrates purity of pegylated L-asparaginase (Example 2) by analytical HP-size exclusion chromatography (HP-SEC).
Figure 3 illustrates purity of pegylated L-asparaginase (Example 3) by analytical HP-size exclusion chromatography (HP-SEC).
Figure 4 illustrates purity of pegylated L-asparaginase (Example 4) by analytical HP-size exclusion chromatography (HP-SEC).
Figure 5 illustrates purity of pegylated L-asparaginase (Example 5) by analytical HP-size exclusion chromatography (HP-SEC).
Figure 6 illustrates purity of pegylated L-asparaginase (Example 6) by analytical HP-size exclusion chromatography (HP-SEC).
Abbreviations:
CBB: Coomassie brilliant blue
HMW: High molecular weight
HP-SEC: High pressure size exclusion chromatography
hrs: Hours
LMW: Low molecular weight
mTorr: Millitorr
ND: Not detected
PEG: Polyethylene glycol
RH: Relative humidity
RI detector: Refractive index detector
SDS-PAGE: Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
UV : Ultra violet
WFI: Water for injection
b-HPCD: Hydroxypropyl -beta-cyclodextrin Definitions
Certain terms are defined herein below in order to better understand the present invention.
The term“L-asparaginase” represents an amidohydrolase which catalyzes L-asparagine into L- aspartic acid and ammonia. It also includes pegylated form of L-asparaginase (pegylated L- asparaginase). Said pegylated form of L-asparaginase includes L-asparaginase which is covalently conjugated to monomethoxypolyethylene glycol (mPEG). Example of pegylated L- asparaginase is pegaspargase and calaspargase sold under brand name Oncaspar® and Asparlas™ respectively.
The term“pharmaceutical” as used herein is with reference to a composition, e.g., an aqueous formulation or a lyophilized formulation that it is useful for treating a disease or disorder.
The term“therapeutic amount” refers to the amount of therapeutic agent used to treat disorder or disease without causing toxicity to the patients. The therapeutic agent according to the present invention is pegylated L-asparaginase or L-asparaginase.
The term“pharmaceutical formulation” refers to preparations, which are in such form as to permit the biological activity of the active ingredients to be unequivocally effective. The term “pharmaceutical formulation” or“pharmaceutical composition” or“composition” can be used here interchangeably.
The“lyophilized formulation” is a dosage form, which is prepared by lyophilization or freeze drying process. The lyophilization was performed with lyophilization technique involving steps such as freezing, primary drying, secondary drying and optionally annealing. According to the present invention freezing is rapid freezing as described herein.
The“frozen composition of L-asparaginase or pegylated L-asparaginase” is a dosage form in which freezing of aqueous solution of L-asparaginase or pegylated L-asparaginase is conducted at or below -15 °C.
The term“excipient” refers to an agent that may be added to a formulation to stabilize the active drug substance in the formulated form to adjust and maintain osmolality and pH of the pharmaceutical preparations. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. “Pharmaceutically acceptable” excipients are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. The term“salt” according to the present invention is a component which is not a part of a buffer system. Preferably, such salts are inorganic salts which is not a part of buffer system. The term “inorganic salt” refers to any salt that does not contain C-H bonds as opposed to organic salt that contains C-H bonds. Inorganic salts such as sodium chloride, potassium chloride etc. are widely used as stabilizers or tonicity agents in addition to their use in a buffer system in the pharmaceutical formulations. Examples of inorganic salts include, but are not limited to, sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCl2), barium chloride (BaCE), ferric chloride (FeCE) etc.
The term“free from salt” according to present invention refers to the composition of pegylated L- asparaginase which does not include salt in addition to buffer. The salt referred in“free from salt” is the salt that has no buffering capacity. According to the present invention, preferably, inorganic salt is not added as an excipient.
The term“buffer” or“buffer solution” or“buffer system” are well known to a person skilled in the art and generally refers to an aqueous solution comprising a mixture of an acid (usually a weak acid, e.g. succinic acid) and its conjugate base (e.g. succinate or sodium succinate) or alternatively a mixture of a base (usually a weak base, e.g. histidine) and its conjugate acid (e.g. protonated histidine salt). A buffered solution prevents change of pH of the solution due to the “buffering capacity” imparted by the“buffering agent(s)”. The pH of a "buffer solution" will change very only slightly upon addition of a small quantity of strong acid or base due to the “buffering effect” imparted by the“buffering agent(s)”.
The term“buffer system” is well known to a person skilled in the art and generally comprises one or more buffering agent(s) and/or an acid/base conjugate(s) thereof, and more suitably comprises one buffering agent only and an acid/base conjugate thereof. Unless stated otherwise, any concentrations stipulated herein in relation to a“buffer system” (i.e. a buffer concentration) suitably refers to the combined concentration of the buffering agent(s) and/or acid/base conjugate(s) thereof. In other words, concentrations stipulated herein in relation to a“buffer system” suitably refer to the combined concentration of all the relevant buffering species (i.e. the species in dynamic equilibrium with one another, e.g. succinate / succinic acid). As such, a given concentration of a succinate buffer system generally relates to the combined concentration of succinate (or succinate salt(s), e.g. sodium succinate) and its corresponding acid. Skilled person can also use other suitable acid for the preparation of buffer system. The overall pH of the composition comprising the relevant buffer system is generally a reflection of the equilibrium concentration of each of the relevant buffering species (i.e. the balance of buffering agent(s) to acid / base conjugate(s) thereof).
The term“buffering agent” is well known to a person skilled in the art and generally refers to an acid or base component (usually a weak acid or weak base) of a buffer or buffer solution. A buffering agent maintain the pH of a given solution at or near to a pre-determined value, and the buffering agents are generally chosen to complement the pre-determined value. A buffering agent is suitably a single compound which gives rise to a desired buffering effect, especially when said buffering agent is mixed with (and suitably capable of proton exchange with) an appropriate amount (depending on the pre-determined pH desired) of its corresponding “acid/base conjugate”, or if the required amount of its corresponding“acid / base conjugate” is formed in- situ which can be achieved by adding strong acid or base until it attains the required pH.
The term“acid / base conjugate” is well known to a person skilled in the art and generally refers to the conjugate acid or conjugate base (whichever is relevant at a particular pH - typically the conjugate acid in the context of the present invention) of a particular "buffering agent". The acid / base conjugate of a succinate buffering agent (e.g. sodium succinate) is suitably succinic acid.
The term “buffering species” refers to the particular species (excluding any associated counteranions or countercations - i.e. ignore sodium ions for sodium succinate / succinic acid systems) of a given buffer system which are in dynamic equilibrium with (and proton-exchange with) one another. For example, succinate anions and succinic acid together constitute the “succinate buffering species” of a“succinate buffer system”.
The term“high molecular weight species” or“low molecular weight species” are product related substances that contributes to the size heterogeneity of a protein. Herein, the present invention protein is pegylated L-asparaginase. High molecular weight species may include dimers, trimers, or aggregates of a desired protein, pegylated and / or non-pegylated form of desired protein, preferably pegylated L-asparaginase, as well as undesired polypeptide contaminants. LMW may include free unmodified asparaginase, depegylated or fragmented asparaginase.
A“disorder” or“disease” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions, which predisposes the subject to the disorder in question. In a pharmacological sense, in the context of the present invention, a“therapeutically effective amount” or“effective amount” of an antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the antibody is effective.
The term“water” is intended to mean water that has been purified to remove contaminants, usually by distillation or reverse osmosis, also referred to herein as“pure water”. In a preferred embodiment, water used in the methods and compositions of the invention is excipient-free. Preferably, water includes sterile water suitable for administration to a subject. In another embodiment, water is meant to include water for injection (WFI).
The terms“patient” and“subject” are used interchangeably and are used in their conventional sense to refer to a living organism suffering from or prone to a condition that can be prevented or treated by administration of a composition of the present invention, and includes both 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 gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, adult, juvenile and newborn individuals are of interest.
Embodiments of the invention
In one embodiment, the invention provides pharmaceutical formulation comprising a therapeutic amount of L-asparaginase.
In second embodiment, the invention provides pharmaceutical formulation comprising a therapeutic amount of pegylated L-asparaginase. In third embodiment, the pharmaceutical formulation of L-asparaginase according to the present invention is a liquid formulation or lyophilized formulation.
In a preferred embodiment, the pharmaceutical formulation of L-asparaginase according to the present invention is a lyophilized formulation. In fourth embodiment, the pharmaceutical formulation of pegylated L-asparaginase according to the present invention is a liquid formulation or lyophilized formulation.
In a preferred embodiment, the pharmaceutical formulation of pegylated L-asparaginase according to the present invention is a lyophilized formulation. In fifth embodiment, the lyophilized formulation according to the present invention comprises L- asparaginase with one or more suitable excipient(s).
In sixth embodiment, the lyophilized formulation according to the present invention comprises pegylated L-asparaginase with one or more suitable excipient(s).
In seventh embodiment, the present invention provides a pharmaceutical formulation suitable for human use which comprises a therapeutic amount of L-asparaginase, sugar and other suitable excipient(s) optionally selected from buffer, amino acid, surfactant, anti-oxidant, anti-microbial agent and WFI.
In a preferred embodiment, the present invention provides a pharmaceutical formulation suitable for human use which comprises L-asparaginase and a suitable sugar. In another embodiment, the present invention provides pharmaceutical formulation of L- asparaginase comprises buffer.
In another embodiments, the pharmaceutical formulation of L-asparaginase according to the present invention comprises one or more amino acid.
In another embodiment, the present invention provides pharmaceutical formulation of L- asparaginase comprises suitable sugar, buffer and optionally one or more amino acid and/or surfactant.
In an embodiment, the present invention provides a pharmaceutical formulation suitable for human use which comprises a therapeutic amount of pegylated L-asparaginase, sugar and other suitable excipient(s) optionally selected from buffer, amino acid, surfactant, anti-oxidant, anti- microbial agent and WFI. In a preferred embodiment, the present invention provides a pharmaceutical formulation suitable for human use which comprises pegylated L-asparaginase and suitable sugar.
In another embodiment, the present invention provides pharmaceutical formulation of pegylated L-asparaginase comprises buffer. In another embodiments, the pharmaceutical formulation of pegylated L-asparaginase according to the present invention comprises one or more amino acid.
In another embodiment, the present invention provides pharmaceutical formulation of pegylated L-asparaginase comprising suitable sugar, buffer and optionally one or more amino acid and/or surfactant.
In yet another embodiment, the method of lyophilizing an aqueous composition that comprises L- sparaginase, a buffer, a sugar optionally an amino acid and/or surfactant in a manner sufficient to produce stable lyophilized L-asparaginase composition.
In an embodiment, the method of lyophilizing an aqueous composition that comprises a pegylated L-asparaginase, a buffer, a sugar optionally an amino acid and / or surfactant in a manner sufficient to produce stable lyophilized pegylated L-asparaginase composition.
In another embodiment, the lyophilized formulation according to the present invention is reconstituted with water.
In one of the embodiments, the pharmaceutical formulation of L-asparaginase is free from salt(s). In a preferred embodiment, the pharmaceutical formulation of L-asparaginase is free from inorganic salt(s). In one of the embodiments, the pharmaceutical formulation of pegylated L-asparaginase is free from salt(s). In a preferred embodiment, the pharmaceutical formulation of pegylated L- asparaginase is free from inorganic salt(s). In a preferred embodiment, the pharmaceutical formulation of L-asparaginase comprising a therapeutic amount of L-asparaginase, sugar and buffer optionally with one or more suitable excipients, wherein the formulation is free from salt. In more preferred embodiment, the pharmaceutical formulation of pegylated L-asparaginase comprising a therapeutic amount of pegylated L-asparaginase, sugar and buffer optionally with one or more suitable excipients, wherein the formulation is free from salt.
In one more embodiment, the aqueous formulation of present invention comprises L-asparaginase in water. In one more embodiment, the aqueous formulation of present invention comprises pegylated L- asparaginase in water.
In one of the embodiments of the invention, the aqueous pharmaceutical formulation according to the present invention further comprises one or more excipients selected from a sugar, buffer, amino acid, surfactant and the like. In one more embodiment, the pharmaceutical formulation of present invention maintains structural integrity of L-asparaginase upon multiple freezing cycles. In a preferred embodiment, the pharmaceutical formulation of present invention maintains structural integrity of L- asparaginase at least after two freeze-thaw cycle, more preferably, at least after five freeze-thaw cycle. In one more embodiment is provided a pharmaceutical formulation of L-asparaginase according to the present invention, wherein L-asparaginase is pegylated L-asparaginase or pegaspargase or calaspargase.
In one more embodiment, the pharmaceutical formulation of present invention maintains structural integrity of pegylated L-asparaginase upon multiple freeze-thaw cycles. In a preferred embodiment, the pharmaceutical formulation of present invention maintains structural integrity of pegylated L-asparaginase at least after two freeze-thaw cycle, more preferably, at least after five freeze-thaw cycle. In one of the embodiments, the pharmaceutical formulation of present invention contains low level of bacterial endotoxins. In a preferred embodiment, the pharmaceutical formulation of present invention contains less than 100 EU / mg bacterial endotoxins.
In one embodiment, the method of preparing lyophilized formulation of the present invention comprises the steps of freezing, primary drying, secondary drying and optionally with the step of annealing. In a preferred embodiment, the present invention provides method of preparing lyophilized formulation of L-asparaginase or pegylated L-asparaginase comprising steps of freezing, primary drying and secondary drying optionally annealing, wherein freezing is performed at a relatively faster rate to achieve the desired freezing temperature.
In a preferred embodiment, method of preparing lyophilized formulation of pegylated L- asparaginase of the present invention comprises following steps:
(a) Freezing at around - 50 °C which is achieved in less than an hour ;
(b) Primary drying cycle, under vacuum at 40 - 60 mTorr for at least 24 hours at a temperature range from about - 50 °C to - 20 °C and
(c) Secondary drying cycle at relatively higher temperature between +20 °C and +30 °C for at least about 24 hours.
Detailed description of the present invention
The present invention provides a stable composition of L-asparaginase, preferably a stable composition of pegylated L-asparaginase. The composition according to the present invention includes one or more of a sugar, buffer and an amino acid as an excipient along with L- asparaginase. The current invention further provides methods of making the composition of L- asparaginase, preferably of pegylated L-asaparaginase. The composition according to the present invention finds therapeutic use in single-dose form or in multi-dose form. The present invention includes stable frozen or lyophilized L- asparaginase composition, preferably pegylated L- asparaginase composition. The frozen or lyophilized composition of L-asparaginase or pegylated L-asaparaginase according to the present invention comprises one or more of a sugar, buffer and an amino acid. The lyophilization process to prepare formulation of the present invention is performed using the techniques available in the art, which includes various steps such as freezing preferably rapid freezing, primary drying, secondary drying and optionally annealing. In one of the aspects, the formulation of the invention has improved stability either in frozen or lyophilized or liquid form.
In a further aspect, the formulation of the invention maintains stability after at least one freeze- thaw cycle.
In further describing the embodiments of the present disclosure, composition (e.g., frozen or lyophilized or liquid) and methods of making composition of present invention are described in details.
The present invention provides stable pharmaceutical composition comprising therapeutic amount of L-asparaginase, preferably therapeutic amount of pegylated L-asparaginase. The pegylated L-asparaginase according to the present invention is an L-asparaginase covalently linked to polyethylene glycol (PEG). The pegylated L-asparaginase may include PEG covalently linked to the asparaginase at one or more positions on the asparaginase. For example, PEG may be covalently linked to an amino acid residue of the asparaginase. In some cases, the PEG is covalently linked to an amine group of an amino acid residue of the asparaginase. In some embodiments, the PEG is covalently linked to an amino acid side chain of an N-terminal amino acid in the asparaginase. In some embodiments, the PEG is covalently linked to an epsilon-amino group of lysine in the asparaginase. In some embodiments, the PEG is covalently linked to an amino acid side chain of the N-terminal amino acid and an epsilon-amine group of lysine(s) in the asparaginase. In some instances, the pegylated L-asparaginase is substantially non-antigenic. In some instances, the pegylated L-asparaginase has a reduced rate of clearance from the systemic circulation of a subject as compared to an unmodified L-asparaginase.
The pharmaceutical formulation of L-asparaginase or pegylated L-asparaginase according to present invention may be administered to the subject in particular dosage. The “dosage” according to the present invention refers to amount of active ingredient administered to the subject in single dose or multiple dose. In one embodiment, the dosage unit includes an amount of pegylated L-asparaginase ranging from 500 to 1,000 IU / mL. In further embodiment, the dosage unit includes an amount of the pegylated L-asparaginase ranging from 700 to 800 IU / mL. In preferred embodiment, the amount of pegylated L-asparaginase in composition according to the present invention is about 750 IU / mL. The term “about” refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods, and other similar considerations. The term“about” also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. The compositions of the present disclosure, in addition to the pegylated L- asparaginase, may include carbohydrate. In one embodiment, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising carbohydrate. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may also be employed. Some carbohydrate excipients of interest include, but are not limited to, for example, monosaccharides, such as fructose, maltose, galactose, glucose, mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. For instance, the composition may include a sugar. Preferred sugars suitable for use in the compositions of the present invention include sugars that are compatible with the L- asparaginase as well as compatible with pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration. Examples of suitable sugars include, but not limited to, sucrose, mannitol, sorbitol, maltose, trehalose, hydroxypropyl-beta- cyclodextrin (b-HPCD), lactose, glucose, fructose, galactose, glucosamine, and the like, and combinations thereof. In certain instances, the sugar is a disaccharide. For example, the disaccharide may be sucrose or trehalose. In some instances, the sugar is a sugar alcohol. For example, the sugar alcohol may be sorbitol or mannitol. In one embodiment, the suitable sugar comprises trehalose, sucrose or mannitol or suitable combinations thereof.
In one embodiment, the present invention provides a composition of F-asparaginase or pegylated F-asparaginase comprising sugar selected from monosaccharides, disaccharides, polysaccharides and alditols.
In further embodiment, the present invention provides a composition of F-asparaginase or pegylated F-asparaginase comprising sugar selected from fructose, maltose, galactose, glucose, mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrins, dextrans, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myoinositol and suitable combinations thereof.
In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar selected from lactose, sucrose, trehalose, sorbitol, mannitol, raffinose, and suitable combinations thereof. In a more preferred embodiment the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar selected from sucrose, trehalose, mannitol and suitable combinations thereof.
In another embodiment, the sugar concentration according to the present invention, is in the range of about 0.05 mg / mL to 150 mg / mL. Preferably, the sugar concentration according to the present invention is in the range of about 0.1 mg / mL to 100 mg / mL. More preferably, the sugar concentration according to the present invention is in the range of about 0.05 mg / mL to 20 mg / mL or about 0.05 mg / mL to 50 mg / mL or about 50 mg / mL to 75 mg / mL or about 50 mg / mL to 100 mg / mL or about 40 mg / mL to 90 mg / mL or 50 mg / mL to 150 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between particular ranges. For Example, the concentration range of sugar about 0.05 mg / mL to 150 mg / mL include 0.05 mg / mL, 0.06 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 5.0 mg / mL, 10.5 mg / mL, 50.0 mg / mL, 60.0 mg / mL, 75.0 mg / mL, 85.0 mg / mL, 100.0 mg / mL, 125.0 mg / mL, 150.0 mg / mL or any integer or non-integer number in between them. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in the range of 40 mg / mL to 90 mg / mL. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in the range of 60 mg / mL to 90 mg / mL. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar in amount of 60 mg / mL or 65 mg / mL or 75 mg / mL or 85 mg / mL or 90 mg / mL. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sucrose at a concentration of about 65 mg / mL or about 75 mg / mL or about 85 mg / mL or about 90 mg / mL. In one more preferred embodiment, the present invention provides a composition of L- asparaginase or pegylated L-asparaginase comprising trehalose at a concentration of about 60 mg / mL. Another excipient that may be included in the compositions of the present disclosure is a buffer. Buffers suitable for use in the compositions of the present disclosure include buffers that are compatible with the L-asparaginase as well as with the pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration. Examples of suitable buffers include, but are not limited to, phosphate buffer, histidine buffer, glycine buffer, arginine buffer, citrate buffer, succinate buffer, acetate buffer, tromethamine buffer, bicarbonate buffer, carbonate buffer, sulfate buffer, chloride buffer and suitable combinations thereof and the like. In one embodiment, the suitable buffer according to the present is selected from phosphate buffer, histidine buffer, glycine buffer, arginine buffer, citrate buffer, succinate buffer, acetate buffer, tromethamine buffer, bicarbonate buffer, carbonate buffer, sulfate buffer, chloride buffer and suitable combinations thereof. In a preferred embodiment, the suitable buffer according to the present invention is selected from phosphate buffer, histidine buffer, succinate buffer and suitable combination thereof. In a preferred embodiment, the suitable buffer according to the present invention is a phosphate buffer or a succinate buffer. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer wherein buffer is succinate buffer or phosphate buffer.
The buffer included in the composition according to the present invention may be a buffer that maintains the pH of the composition at a pH, ranging from about pH 5.0 to about pH 8.0, or a pH of about 7.0. The pH range 5.0 to 8.0 according to the present invention includes pH 5.0, pH 6.0, pH 7.0 or pH 8.0 or any non-integer number in between them. In one of the embodiments, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH in the range of pH 5.0 to pH 8.0. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH in the range of pH 6.0 to pH 7.5. pH 6.0 to pH 7.5 according to the present invention includes pH 6.0, pH 6.5, pH 7.0, pH 7.3, pH 7.5 or any non-integer or integer number in between them. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase having a pH 7.3 or pH 6.5.
In one embodiment, the buffer concentration according to the present invention is in the range of about 5 mM to 100 mM. In a preferred embodiment, the buffer concentration is in the range of about 5 mM to 75 mM. The concentration range of buffer according to present disclosure include each integer and non-integer number between particular range. Lor example, the concentration range of about 5 mM to 100 mM include 5.0 mM or 10.0 mM or 20.0 mM or 30.0 mM or 40.0 mM or 50.0 mM or 60.0 mM or 70.0 mM or 80 mM or 90 mM or 100 mM or any integer or non integer number in between them. The concentration range of buffer in millimolar (mM) as mentioned above according to the present invention includes corresponding equivalent concentration in mg / mL. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer in the range of about 5 mM to about 49 mM. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising buffer at a concentration, 10 mM or 25 mM or 49 mM. In one more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising phosphate buffer at a concentration, 25 mM or 49 mM. In one more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising succinate buffer at a concentration, 10 mM.
Another excipient that may be included in the compositions of the present disclosure is an amino acid. Amino acids suitable for use in the compositions of the present disclosure include amino acids that are compatible with the L-asparaginase as well as with the pegylated L-asparaginase and suitable for administration to a subject, e.g., by injection or intravenous administration. Examples of suitable amino acids include, but are not limited to, arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine / cystine and the like, their suitable salt form or suitable combinations of any of the above. In one embodiment, the suitable amino acid is selected from arginine, glycine, leucine, lysine, histidine, methionine, glutamic acid or their suitable salt form or combinations, thereof. Amino acid according to the present invention can be a part of buffer as a buffering agent or buffering species or acid / base conjugate. In one of the embodiments, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising amino acid selected from arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine / cysteine, their suitable salt form and combination thereof. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising amino acid selected from arginine, glycine, leucine, lysine, histidine, methionine, glutamic acid, their suitable salt form and suitable combinations thereof. In a more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising L-arginine or glycine or suitable combinations thereof.
In another embodiment, the amino acid according to the present invention is present in amount about 0.1 mg / mL to about 100 mg / mL, preferably the amino acid is present in amount about 0.1 mg / mL to about 50 mg / mL, more preferably the amino acid is present in amount about 0.1 mg / mL to about 10 mg / mL. The concentration range according to present disclosure include each integer and non-integer number between a particular range. For Example, the concentration range of about 0.1 mg / mL to about 100 mg / mL include 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 5.0 mg / mL, 10.5 mg / mL, 50.0 mg / mL, 75.0 mg / mL, 100.0 mg / mL or any integer or non-integer number in between them. In one of the embodiments, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising amino acid in the range of 0.1 mg / mL to 100 mg / mL, preferably 0.5 mg / mL to 10.0 mg / mL. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising L- arginine at a concentration 5 mg / mL. In a one more preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L- asparaginase comprising glycine at a concentration 5 mg / mL.
In a one of the embodiments, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising a sugar, a buffer and optionally an amino acid, in addition to the pegylated L-asparaginase. In a preferred embodiment, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising sugar and buffer in addition to the pegylated L-asparaginase. In a more preferred embodiment, the present invention provides a lyophilized composition of L-asparaginase or pegylated L-asparaginase comprising sugar and buffer in addition to the pegylated L-asparaginase. For example, preferred aspects of the present disclosure include a lyophilized storage stable composition of a pegylated L- asparaginase comprises suitable amount of trehalose or sucrose or raffinose or sorbitol or lactose or mannitol as a sugar, succinate or phosphate or histidine as a buffer and optionally arginine or glycine or methionine or histidine or glutamic acid as an amino acid.
In a preferred embodiment, the present invention provides a stable lyophilized composition of L- asparaginase or pegylated L-asparaginase comprising pegylated L- asparaginase, sugar and buffer. In a more preferred embodiment, the present invention provides a stable lyophilized composition comprising pegylated L- asparaginase, sucrose or trehalose, phosphate buffer or succinate buffer optionally with one or more excipients as described in the specification.
Compositions of the present disclosure may also include other components, such as additional pharmaceutically acceptable excipients or a dosage delivery vehicle as part of the composition. Additional excipients according to the present invention may include, but are not limited to, antimicrobial agents, anti-oxidants, surfactants, water (e.g., water for injection (WFI)), alcohols, glycerin, vegetable oils, phospholipids and any combinations thereof.
Examples of surfactants according to the present invention include, but not limited to polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (e.g. Brij), alkylphenylpolyoxyethylene ethers (e.g. Triton-X), polyoxyethylene -polyoxypropylene copolymer (e.g. Poloxamer, Pluronic), sodium dodecyl sulphate (SDS) and the like. Preferably, the suitable surfactant is polyoxyethylensorbitan-fatty acid esters (Tweens). More preferably, the polyoxyethylensorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™), polysorbate 80 (sold under the trademark Tween 80™) and polyethylene -polypropylene copolymers which are sold under the names Pluronic (R) F68 or Poloxamer 188™.
In one of the preferred embodiments, the present invention provides a composition of L- asparaginase or pegylated L-asparaginase free from salt. In more preferred embodiments, the present invention provides a composition of L-asparaginase or pegylated L-asparaginase comprising L-asparaginase or pegylated L-asparaginase as an active ingredient, sugar, buffer and wherein the composition is free from salt. The salt according to the present invention which is not present may be selected from sodium chloride (NaCl), potassium chloride (KC1), calcium chloride (CaCL), barium chloride (BaCL), ferric chloride (FeCF). Composition of pegylated L- asparaginase known in the art includes salt in addition to buffer and sugar while the composition of pegylated L-asparaginase according to the current invention does not include salt in addition to buffer. According to the present invention, salts, that have no buffering capacity, are not added in composition of pegylated L-asparaginase. The composition according to the present invention is stable and can provide long-term stability. It is illustrated herein below examples by providing analytical data of stability analysis at various temperature and at various forms of composition by HP-SEC.
In certain instances, the composition (e.g., frozen or lyophilized or liquid) is a sterile composition. By“sterile” is meant that there are substantially no microbial contaminants (e.g., fungi, bacteria, viruses, spore forms, etc.). In one instance, the composition (e.g., frozen or lyophilized or liquid) contains low level of bacterial endotoxins, preferably less than 100 EU / mg.
The present invention provides pharmaceutical composition of pegylated L-asparaginase that is substantially free of particles and/or aggregates and prevents the formation of particles and/or aggregates during storage and/or transport. The present invention provides pharmaceutical composition of pegylated L-asparaginase that is stable for at least one month at storage condition between +2 °C and +8 °C, at least one month at storage condition of 30 °C ± 2 °C and RH 65 % ± 5 and at least one month at storage condition of 40 °C ± 2 °C and RH 75 % ± 5. It is illustrated herein below examples by providing analytical data of stability analysis at various temperature and at various forms of composition by HP-SEC. Such stable formulation is prepared with specific lyophilization process as described in this application.
Method of preparing lyophilized formulation comprising freezing preferably rapid freezing, annealing, primary drying and secondary drying is performed preferably in 40 - 120 hours. Pegylated L-asparaginase solution filled in USP Type I glass vials is lyophilized after loading the vials in a lyophilizer, from ambient temperature to +5 °C, followed by freezing at around - 50 °C which is achieved in less than an hour, at a relatively faster rate than the process known in the art for lyophilization of L-asparaginase or pegylated L-asparaginase. Upon freezing, the frozen mass in the vial is held for another at least 1 hour at the same temperature and undergoes primary drying cycle, under vacuum at 40 - 60 mTorr for at least 24 hours at a temperature range from about - 50 °C to - 20 °C. Lollowing primary drying, secondary drying cycle is carried out at relatively higher temperature between +20 °C and +30 °C for at least about 24 hours. At the end of lyophilization cycles, vials are fully stoppered under No and unloaded at ambient pressure and temperature.
Analytical methods used in the present invention:
Clarity and Color:
Samples were analyzed for clarity and color as per Ph. Eur. 2.2.1 and 2.2.2 respectively.
pH:
Samples were analyzed for pH by potentiometric technique. HP-Size exclusion
Figure imgf000022_0001
(HP-SEC) and estimation of free PEG:
Samples were analyzed to determine the purity, level of HMW and LMW species in pegylated L- asparaginase preparations by HP-SEC using YMC Pak Diol-300 column (8.0 mm I.D x 30 cm L). Samples containing pegylated L- asparaginase were loaded and eluted isocratic in mode using sodium phosphate buffer containing 2-propranol (90: 10 v/v) at a flow rate of 0.5 mL / min. Elution was monitored at UV 214 nm for the protein peaks. Free PEG was determined with RI detection.
SDS-PAGE:
Samples were analyzed by denaturing gel electrophoresis under non-reducing conditions on 4 - 20 % polyacrylamide gel and detection of pegylated protein performed by using coomassie brilliant blue solution R250 staining solution.
Enzyme activity:
L-asparaginase activity in pegylated form was determined by estimating the ability of the enzyme to effectively convert the substrate, L-asparagine, to aspartate and ammonia. The quantitative determination of the released ammonia directly correlates with the effective amount of active enzyme present in pegylated L-asparaginase.
Protein concentration by method:
Figure imgf000022_0002
The UV absorbance of pegylated L-asparaginase samples were measured at 280nm in 1 cm path length on UV spectrophotometer to check the concertation of pegylated L-asparaginase. The respective formulation buffer was used as blank.
These and other advantages of the invention, as well as additional inventive features, will be apparent from the descriptions of the invention provided, herein.
Examples
The following non-limiting examples describe the different formulations, which can be prepared as per the present invention. It will be appreciated that other excipients may be added as are necessary to these formulations and such addition of excipients are within the scope of a person skilled in the art and are to be included within the scope of the present invention.
Example 1
Figure imgf000023_0001
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration(s), as described above. pH of the formulation medium was maintained at around pH 7.30. Excipient(s) were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with sterile water or Water for Injection. The formulated pegylated L-asparaginase was evaluated for stability upon repeated freezing and thawing as shown in Table 13. Formulated samples were analyzed for purity by HP- SEC. Purity of pegylated L-asparaginase sample after formulation was found to be 99.81 % as shown in Figure 1. A person skill in the art can also formulate the composition for pegylated L- asparaginase and can fill in the suitable container-closure system (like vials, cartridges, syringes etc.) for storage. Lyophilization of the formulated sample was carried out in the above specified container-closure systems. Lyophilization was performed in about 110 hours. Pegylated L- asparaginase solution filled in USP Type I glass vials was lyophilized after loading the vials in a lyophilizer, from ambient temperature to +5 °C, followed by freezing at around - 45 °C which is achieved in less than an hour. Upon freezing, the frozen mass in the vial was held for another 2 hours at the same temperature and subjected to primary drying cycle, under vacuum at about 50 mTorr for about 65 hours at a temperature range from about - 45 °C to - 20 °C. Following primary drying, secondary drying cycle was carried out at relatively higher temperature about +25 °C for about 40 hours. At the end of lyophilization cycles, vials were fully stoppered under
No and unloaded at ambient pressure and temperature.
A person skilled in the art can further evaluate the stability of formulated pegylated L- asparaginase at various temperature conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed storage conditions (higher temperature; about +40 °C). Example 2
Figure imgf000024_0001
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose and L- arginine at desired concentration(s), as described above. pH of the formulation medium was maintained at around pH 7.30. Excipient(s) were added to the protein solution from respective stock solutions to adjust the final concentration and volume was made up to the desired level with sterile water or Water for Injection. The formulated pegylated L-asparaginase was evaluated for stability upon repeated freezing and thawing as shown in Table 13. Formulated samples were analyzed for purity by HP-SEC. Purity of pegylated L-asparaginase sample after formulation was found to be 99.81 % as shown in Figure 2. A person skill in the art can also formulate the composition for pegylated L-asparaginase and can fill in the suitable container-closure system (like vials, cartridges, syringes etc.) for storage. Lyophilization of the formulated sample was performed as described in example 1. A person skilled in the art can further evaluate the stability of formulated pegylated L-asparaginase at various temperature conditions like Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (about +25 °C) or stressed storage conditions (higher temperature; about +40 °C).
Example 3
Figure imgf000025_0001
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 7.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed with as described in example 1. Table 1 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 3. Purity of pegylated L-asparaginase after formulation was found to be 99.13 %, as shown in Figure 3.
Stability of lyophilized pegylated L-asparaginase prepared with example 3 was evaluated over a period of 30 days (1 month), under various storage conditions, such as Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (30 °C ± 2 °C; RH 65 % ± 5 %), and Stressed temperature condition (40 °C ± 2 °C; RH 75 % ± 5 %). Lyophilized samples exposed to different temperature conditions were withdrawn at different time points, reconstituted and analyzed for purity by HP-SEC, as indicated in Table 2 (Real-Time), Table 3 (Accelerated) and Table 4 (Stressed). Table 5 illustrates the stability (in-use) of reconstituted pegylated L-asparaginase stored between +2 °C and +8 °C, as assessed by HP-SEC for purity.
Table 1: Results obtained for physicochemical properties and enzyme activity of pegylated
L-asparaginase prepared with example 3
Figure imgf000026_0001
Table 2: Purity of lyophilized pegylated L-asparaginase stored under Real-Time condition as assessed by HP-SEC - Example 3
Figure imgf000026_0002
Table 3: Purity of lyophilized pegylated L-asparaginase stored under Accelerated condition as assessed by HP-SEC - Example 3
Figure imgf000027_0001
Table 4: Purity of lyophilized pegylated L-asparaginase stored under Stressed condition as assessed by HP-SEC - Example 3
Figure imgf000027_0002
Table 5: Stability of reconstituted pegylated L-asparaginase stored between +2 °C and +8
°C as assessed by HP-SEC - Example 3
Figure imgf000027_0003
Example 4
Figure imgf000027_0004
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in succinate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 6.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 6 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 4. Purity of pegylated L- asparaginase after formulation was found to be 99.12 %, as shown in Figure 4.
Stability of lyophilized pegylated L-asparaginase prepared with example 4 was evaluated over a period of 30 days (1 month), under various storage conditions, such as Real-Time storage condition (between +2 °C and +8 °C), Accelerated storage condition (30 °C ± 2 °C; RH 65 % ± 5 %), and Stressed temperature condition (40 °C ± 2 °C; RH 75 % ± 5 %). Lyophilized samples exposed to different temperature conditions were withdrawn at different time points, reconstituted and analyzed for purity by HP-SEC, as indicated in Table 7 (Real-Time), Table 8 (Accelerated) and Table 9 (Stressed). Table 10 illustrates the stability (in-use) of reconstituted pegylated L-asparaginase stored between +2 °C and +8 °C, as assessed by HP-SEC for purity.
Table 6: Results obtained for physicochemical properties and enzyme activity of pegylated
L-asparaginase prepared with example 4
Figure imgf000028_0001
Figure imgf000029_0001
Table 7: Purity of lyophilized pegylated L-asparaginase stored under Real-Time condition as assessed by HP-SEC - Example 4
Figure imgf000029_0002
Table 8: Purity of lyophilized pegylated L-asparaginase stored under Accelerated condition as assessed by HP-SEC - Example 4
Figure imgf000029_0003
Table 9: Purity of lyophilized pegylated L-asparaginase stored under Stressed condition as assessed by HP-SEC - Example 4
Figure imgf000029_0004
Table 10: Stability of reconstituted pegylated L-asparaginase stored between +2 °C and +8
°C as assessed by HP-SEC - Example 4
Figure imgf000030_0001
In summary, the lyophilized formulations, described with Example 3 and Example 4 were found to be stable under various storage conditions, as assessed by HP-SEC for purity of pegylated L- asparaginase.
Example 5
Figure imgf000030_0002
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in phosphate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 7.30. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 11 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 4. Purity of pegylated L- asparaginase after formulation was found to be 99.22 %, as shown in Figure 5. Table 11: Results obtained for physicochemical properties and enzyme activity of pegylated
L-asparaginase prepared with example 5
Figure imgf000031_0001
Example 6
Figure imgf000031_0002
Pegylated L-asparaginase was prepared as per the general process described in patent document WO 1990/013540. It was formulated in succinate buffer, further comprising sucrose at desired concentration, as described above. pH of the formulation medium was maintained at about pH 6.50. The formulated pegylated L-asparaginase was lyophilized in USP Type I glass vial with 20 mm slotted rubber stopper. Lyophilization of the formulated sample was performed as described in example 1. Table 12 illustrates the results of analysis obtained for physicochemical properties and enzyme activity of pegylated L-asparaginase prepared with example 6. Purity of pegylated L- asparaginase after formulation was found to be 99.26 %, as shown in Figure 6.
Table 12: Results obtained for physicochemical properties and enzyme activity of pegylated
L-asparaginase prepared with example 6
Figure imgf000032_0001
Following similar process mentioned in above examples, various other formulation compositions of pegylated L-asparaginase were prepared as summarized herein with examples 7 to 17 shown in Table 13.
Table 13: Various other formulation compositions of pegylated L-asparaginase
Figure imgf000033_0001
Figure imgf000034_0001
Formulations as mentioned in Example 1, Example 2 and Examples 7 - 17, as prepared herein and shown above were exposed to repeated freeze-thaw cycles (5 cycles) to check stability of pegylated L-asparaginase upon freezing and thawing. Samples after repeated freezing and thawing were analyzed for purity by HP-SEC. Results obtained with pegylated L-asparaginase formulated samples after the Ist and 5th freeze-thaw cycles are shown in Table 14. None of the formulation composition was found to show any significant increase in HMW or LMW species. All samples were observed to show greater than 99 % purity even after the 5th freeze-thaw cycle.
Table 14: Results of repeated freeze-thaw analysis of pegylated L-asparaginase by HP-SEC
Figure imgf000035_0001
Figure imgf000036_0001
Incorporation by reference
The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes. Equivalents
The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

We claim:
1. A pharmaceutical formulation of pegylated L-asparaginase comprising a therapeutic amount of pegylated L-asparaginase, sugar and buffer optionally with one or more suitable excipients, wherein the formulation is free from salt.
2. The pharmaceutical formulation as claimed in claim 1 wherein the salt is selected from sodium chloride, potassium chloride, calcium chloride, barium chloride, ferric chloride.
3. The pharmaceutical formulation as claimed in claim 1, wherein the sugar is selected from fructose, maltose, galactose, glucose, mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrins, dextrans, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol, pyranosyl sorbitol, myoinositol and combination thereof.
4. The pharmaceutical formulation as claimed in claim 3, wherein the sugar is selected from lactose, sucrose, trehalose, sorbitol, mannitol, raffinose and combination thereof, preferably selected from sucrose, mannitol and trehalose.
5. The pharmaceutical formulation as claimed in claim 4, wherein the concentration of sugar is in the range of about 0.05 mg / mL to 150 mg / mL, preferably, in the range of about 0.1 mg / mL to 100 mg / mL, more preferably, in the range of 40 mg / mL to 90 mg / mL.
6. The pharmaceutical formulation as claimed in claim 5, wherein sugar is present in amount of 60 mg / mL or 65 mg / mL or 75 mg / mL or 85 mg / mL or 90 mg / mL.
7. The pharmaceutical formulation as claimed in claim 1, wherein the buffer is selected from phosphate buffer, histidine buffer, glycine buffer, arginine buffer, citrate buffer, succinate buffer, acetate buffer, tromethamine buffer, bicarbonate buffer, carbonate buffer, sulfate buffer, chloride buffer and combinations thereof.
8. The pharmaceutical formulation as claimed in claim 7, wherein the buffer is selected from phosphate buffer, histidine buffer, succinate buffer and combination thereof.
9. The pharmaceutical formulation as claimed in claim 8, wherein the concentration of buffer is in the range of about 5 mM to 100 mM, preferably, in the range of about 5 mM to 75 mM, more preferably in the range of about 5 mM to 49 mM.
10. The pharmaceutical formulation as claimed in claim 1 wherein suitable excipient is selected from amino acid, surfactant, anti-oxidant, anti microbial agent, WFI and/or combination thereof.
11. The pharmaceutical formulation as claimed in claim 10, wherein the amino acid is selected from arginine, glycine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, cysteine / cysteine, their suitable salt form and combination thereof, preferably selected from arginine, glycine, leucine, lysine, histidine, methionine, glutamic acid, their suitable salt form and combination thereof.
12. The pharmaceutical formulation as claimed in claim 11, wherein the amino acid is present in the amount of about 0.1 mg / mL to about 100 mg / mL, preferably in the amount of about 0.1 mg / mL to about 50 mg / mL, more preferably in the amount of about 0.1 mg / mL to about 10 mg / mL.
13. The pharmaceutical formulation as claimed in any preceding claim has a pH in the range of pH 5.0 to pH 8.0, preferably pH 6.0 to pH 7.5.
14. The pharmaceutical formulation as claimed in claim 1, wherein the amount of L-asparaginase is ranging from 700 to 800 IU / mL, preferably the amount of L-asparaginase is about 750 IU / mL.
15. The pharmaceutical formulation as claimed in any preceding claims is a liquid formulation or lyophilized formulation, preferably a lyophilized formulation.
16. The pharmaceutical formulation as claimed in any of preceding claims maintains structural integrity of L-asparaginase upon multiple freeze-thaw cycles, preferably at least after two freeze-thaw cycle, more preferably, at least after five freeze-thaw cycle.
17. The pharmaceutical formulation as claimed in any of preceding claims contains low level of bacterial endotoxins, preferably contains less than 100 EU / mg bacterial endotoxins.
18. The lyophilized formulation as claimed in claim 15 is prepared by a method comprising following steps: (a) Rapid freezing, (b) Primary drying, (c) Secondary drying and optionally (d) Annealing.
19. The method of preparing lyophilized formulation as claimed in claim 18 comprising following steps:
(a) Freezing at around - 50 °C which is achieved in less than an hour ;
(b) Primary drying cycle, under vacuum at 40 - 60 mTorr for at least 24 hours at a temperature range from about - 50 °C to - 20 °C and
(c) Secondary drying cycle at relatively higher temperature between +20 °C and +30 °C for at least about 24 hours.
20. The pharmaceutical formulation of pegylated L-asparaginase as claimed in any preceding claim is selected from L- asparaginase, pegaspargase and calaspargase.
PCT/IB2019/059119 2018-11-02 2019-10-24 Pharmaceutical composition of pegylated l-asparaginase WO2020089743A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990013540A1 (en) 1989-04-19 1990-11-15 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
WO1997004801A1 (en) * 1995-07-27 1997-02-13 Genentech, Inc. Stabile isotonic lyophilized protein formulation
WO2018017190A2 (en) 2016-06-01 2018-01-25 Baxalta Incorporated Formulations of polyalkylene oxide-asparaginase and methods of making and using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990013540A1 (en) 1989-04-19 1990-11-15 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
WO1997004801A1 (en) * 1995-07-27 1997-02-13 Genentech, Inc. Stabile isotonic lyophilized protein formulation
WO2018017190A2 (en) 2016-06-01 2018-01-25 Baxalta Incorporated Formulations of polyalkylene oxide-asparaginase and methods of making and using the same

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