Stable liquid formulation of hyaluronidase

Abstract

The present invention provides stable liquid pharmaceutical formulation of Hyaluronidase comprising buffering agent, stabilizer / bulking agent, antioxidant, salt, and optionally, a free amino acid or cryoprotectant. The present liquid pharmaceutical composition is free of surfactant.

Description

[0001] STABLE LIQUID FORMULATION OF HYALURONIDASE

[0002] RELATED APPLICATIONS

[0003] This application is related to Indian provisional application IN202521027887 filed on 25th Mar. 2025 and is incorporated herein in its entirely.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a stable liquid pharmaceutical composition of Hyaluronidase enzyme.

[0006] BACKGROUND OF THE INVENTION

[0007] Recombinant human hyaluronidase (rHuPH20) is a glycosylated single chain protein with 447 amino acids and with calculated molecular weight of full-length polypeptide is 51106 Dalton and with glycosylated molecular weight is 60000-65000 Da.

[0008] There are six N-linked (47, 131, 200, 219, 333, 358) and one O-linked glycosylation sites and 12 cysteines with 6 Di-sulfide linkages which makes it a complex during purification.

[0009] The Hyaluronidase Enzymes are unstable in aqueous systems at room temperature and so are typically stored either in a frozen state at -20° C (-70° C. in some cases), or liquid in the presence of stabilizers such as poly that have low freezing points and low vapour pressures.

[0010] Even under these storage conditions, repeated freeze - thaw and handling at room temperature can lead to loss of activity. The stabilization of enzymes involves the prevention of irreversible protein denaturation / degradation which may occur for a variety of reasons including thermal and others.

[0011] Room Temperature (22-28°C): At this temperature, hyaluronidase experiences gradual denaturation / degradation, especially if it is not protected by stabilizers. The enzyme is most susceptible to thermal degradation in this range.

[0012] Refrigeration (2-8°C): Although refrigeration can slow down the denaturation / degradation process, it is not sufficient to completely prevent loss of enzyme activity, particularly over extended periods. However, stabilizers like sugars and amino acids may improve its shelflife under these conditions.

[0013] Freezing (-20°C): Freezing is a common practice for enzyme storage, but it poses challenges in terms of ice formation, which can disrupt protein structure. Stabilizers such as glycerol, trehalose, and other cryoprotectants can mitigate this effect.Deep freezing (-80°C): Ultra-low temperatures significantly reduce enzyme activity loss by halting most enzymatic processes. This storage condition is optimal for long-term preservation, especially when combined with stabilizers to prevent damage from freezing and thawing cycles.

[0014] Hyaluronidases are a family of enzymes that degrade hyaluronic acid (also known as hyaluronan or hyaluronate), an essential component of the extracellular matrix and a major constituent of the interstitial barrier. By catalysing the hydrolysis of hyaluronic acid, hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. As such, hyaluronidase have been used, for example, as a spreading or dispersing agent in conjunction with other agents, dmgs, and proteins to enhance their dispersion and delivery. Hyaluronidase also have other therapeutic and cosmetic uses. Because of the increasing use of hyaluronidase for therapeutic and cosmetic uses, there is a need for large-scale quantities of purified hyaluronidase. Therefore, among the objects herein, it is an object to provide composition for the stabilization of hyaluronidase.

[0015] According to the present invention, a novel formulation composition without use of human serum albumin as bulk stabilizer is provided. In prior art recombinant hyaluronidase has been stabilized using human serum albumin at very low concentrations of hyaluronidase at below -70°C. The HYLENEX recombinant (hyaluronidase human injection) supplied as sterile, clear, colourless, nonpreserved and ready for use solution. Each mL contains 150 USP units of recombinant human hyaluronidase per mL with 8.5 mg Sodium Chloride, 1.4 mg Dibasic Sodium Phosphate, 1.0 mg Albumin Human, 0.9 mg Edetate Disodium, 0.3 mg Calcium Chloride and Sodium Hydroxide added for pH adjustment.

[0016] W02004078140 discloses a Neutral- Active, Soluble Hyaluronidase Glycoproteins (sHASEGP), portions thereof, particularly Hyaluronidase domains.

[0017] WO2011012637 discloses a highly concentrated, stable pharmaceutical formulation of a pharmaceutically active anti-HER2 antibody comprising: a. about 50 to 350 mg / ml anti-HER2 antibody; b. about 1 to 100 mM of a buffering agent providing a pH of 5.5 ± 2.0; c. about 1 to 500 m of a stabilizer or a mixture of two or more stabilizers; d. about 0.01 to 0.08 % of a nonionic surfactant; and e. an effective amount of at least one hyaluronidase enzyme.

[0018] W02023075506 discloses a pharmaceutical composition comprising (a) a drug, (b) a hyaluronidase, and (c) a poloxamer series surfactant, wherein the hyaluronidase comprises wild-type PH20 having the sequence of SEQ ID NO: 1 or a PH20 variant thereof.

[0019] A formulation combination with increased hyaluronidase stability may help to keep enzyme for long term. Hence, it is necessary to develop a formulation for hyaluronidase enzyme with high stability. The present invention focus on developing a cost effective and high stability formulation of Hyaluronidase enzyme.OBJECTS OF THE INVENTION

[0020] The main object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) buffer; b) sugar / bulking agent; c) antioxidant; d) salt; and e) optionally, a free amino acid or cryoprotectant.

[0021] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; and e) optionally, Arginine or Glycerol.

[0022] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; e) Arginine; and f) Glycerol; wherein said formulation is surfactant-free.

[0023] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; and e) optionally, ImM to 60mM Arginine or 0.5% to 20% Glycerol; wherein said formulation has pH of about 6 to 8 and surfactant free.

[0024] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) about 10 mM Histidine buffer; c) about 119 mM Trehalose; d) about 10 mM Methionine; e) about 100 mM NaCl; and f) optionally, about 20 mM L-Arginine or about 10% Glycerol; wherein the said formulation has pH of about 6.5 to 7.5.

[0025] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) 0.1 to 10 mg / mL Histidine buffer; c) 1 to 150 mg / mL Trehalose; d) 0.1 to 10 mg / mL Methionine; e) 1 to 20 mg / mL Sodium Chloride; and f) optionally, 0.174 to 10.452 mg / mL Arginine or 5 to 200 mg / mL Glycerol; wherein said formulation has pH of about 6 to 8.

[0026] Another object of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) ImM to 50mM Histidine buffer; c) 50mM to 160mM Trehalose; d) ImM to 50mM Methionine; e) 50mM to 150mM NaCl; f) ImM to 60mM Arginine; and g) 0.5% to 20% Glycerol; wherein said formulation has pH of about 6.5 to 7.5.

[0027] SUMMARY OF THE INVENTION

[0028] The main aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) buffer; b) sugar / bulking agent; c) antioxidant; d) salt; and e) optionally, a free amino acid or cryoprotectant.Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; and e) optionally, Arginine or Glycerol.

[0029] Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; e) Arginine; and f) Glycerol; wherein said formulation is surfactant-free.

[0030] Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; and e) optionally, ImM to 60mM Arginine or 0.5% to 20% Glycerol; wherein said formulation has pH of about 6 to 8 and surfactant free.

[0031] Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) about 10 mM Histidine buffer; c) about 119 mM Trehalose; d) about 10 mM Methionine; e) about 100 mM NaCl; and f) optionally, about 20 mM L-Arginine or about 10% Glycerol; wherein said formulation has pH of about 6.5 to 7.5.

[0032] Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) 0.1 to 10 mg / mL Histidine buffer; c) 1 to 150 mg / mL Trehalose; d) 0.1 to 10 mg / mL Methionine; e) 1 to 20 mg / mL Sodium Chloride; and f) optionally, 0.174 to 10.452 mg / mL Arginine or 5 to 200 mg / mL Glycerol; wherein said formulation has pH of about 6 to 8.

[0033] Another aspect of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) ImM to 50mM Histidine buffer; c) 50mM to 160mM Trehalose; d) ImM to 50mM Methionine; e) 50mM to 150mM NaCl; f) ImM to 60mM Arginine; and g) 0.5% to 20% Glycerol; wherein said formulation has pH of about 6.5 to 7.5.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] In order that disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

[0036] Figure 1: Percentage purity (RP-HPLC) analysis of Fl on Day 0

[0037] Figure 2: Percentage purity (RP-HPLC) analysis of Fl on Day 7 at 2-8°CFigure 3: Percentage purity (RP-HPLC) analysis of Fl on Day 7 at -80°C Figure 4: Percentage purity (RP-HPLC) analysis of Fl on Day 30 at 2-8°C Figure 5: Percentage purity (RP-HPLC) analysis of Fl on Day 60 at 2-8°C Figure 6: Percentage purity (RP-HPLC) analysis of Fl on Day 30 at -80°C Figure 7: Percentage purity (RP-HPLC) analysis of Fl on Day 90 at 2-8°C Figure 8: Percentage purity (RP-HPLC) analysis of F2 on Day 60 at 2-8°C Figure 9: Percentage purity (RP-HPLC) analysis of F2 on Day 90 at 2-8°C Figure 10: Percentage purity (RP-HPLC) analysis of F3 on Day 60 at 2-8°C Figure 11: Percentage purity (RP-HPLC) analysis of F3 on Day 90 at 2-8°C Figure 12: Percentage purity (RP-HPLC) analysis of F4 on Day 90 at 2-8°C Figure 13: Representative profile of SE-HPLC purified Hyaluronidase Figure 14: Overlay 10-day room temperature samples

[0038] Figure 15: Comparative assessment of Fl on Day 0, 7 & 30 at -80 °C

[0039] Figure 16: Comparative assessment of Fl on Day 0, 7 & 30 at 2-8 °C

[0040] Figure 17: Comparative assessment of Fl and F3 at -80 °C

[0041] Figure 18: Comparative assessment of Fl and F3 at 2-8 °C

[0042] Figure 19: Comparative assessment of Fl and F2 at -80 °C

[0043] Figure 20: Comparative assessment of Fl and F2 at 2-8 °C

[0044] Figure 21: Comparative assessment of Fl and F4 at -80 °C

[0045] Figure 22: Comparative assessment of Fl and F4 at 2-8 °C

[0046] Figure 23: Stability data RT (-20°C) SEC-HMW% Trend

[0047] Figure 24: Stability dataRT (-20°C) SEC-Monomers% (should be > 90.00 %) Trend Figure 25: Stability data RT (-20°C) pH Trend

[0048] Figure 26: Stability data RT (-20°C) Osmolality Trend

[0049] Figure 27: Stability data RT (-20°C) Trend (Protein Concentration & Purity) Figure 28: Stability data RT (-20°C) Main peak TrendFigure 29: Stability data RT (-20°C) Impurity Trend

[0050] Figure 30: Stability data RT (-20°C) Enzyme Activity Trend

[0051] Figure 31: Stability data 2-8°C SEC-HMW% Trend

[0052] Figure 32: Stability data AT_2-8°C SEC-Monomers% (should be > 90.00 %) Trend

[0053] Figure 33: Stability data AT_2-8°C pH Trend

[0054] Figure 34: Stability data AT_2-8°C Osmolality Trend

[0055] Figure 35: Stability data AT_2-8°C Trend (Protein Concentration & Purity)

[0056] Figure 36: Stability data AT_2-8°C Main peak Trend

[0057] Figure 37: Stability data AT_2-8°C Impurity Trend

[0058] Figure 38: Stability data AT_2-8°C Enzyme Activity Trend

[0059] In the above figures Fl, F2, F3 & F4 are mentioned as below:

[0060]

[0061] DETAILED DESCRIPTION OF THE INVENTION

[0062] The following is a detailed description of embodiments of the invention. The embodiments are in such details as to clearly communicate the invention. However, the amount of details offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents of embodiments, and alternative falling within the spirit and scope of the present invention.

[0063] DEFINITION

[0064] The following definitions are provided to facilitate understanding of certain terms used throughout the specification.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs.Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of particular embodiments, preferred embodiments of compositions, methods and materials are described herein. For the purposes of the present disclosure, the following terms are defined below.

[0066] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0067] The term "about" as used in the present patent specification is meant to specify that the specific value provided may vary to a certain extent.

[0068] The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using "comprising" language, under other circumstances, a related embodiment is also intended to be interpreted and described using "consisting of or "consisting essentially of language.

[0069] As used herein the term "buffering agent providing a pH of 7.0 ± 1.0" refers to an agent which provides that the solution comprising it resists changes in pH by the action of its acid / base conjugate components. The buffer used in the formulations in accordance with the present invention has a pH in the range from about 6.0 to about 8.0, or from about 6.2 to about 7.5, or from about 6.5 to about 7.3. A pH of about 7.0 has to be found to be most suitable. Examples of buffering agents that will control the pH in this range include histidine, acetate, succinate, gluconate, citrate, glycine and other organic acid buffers. The most suitable buffer in accordance with the present invention is a histidine buffer.

[0070] A “Stabilizer / Bulking agent” herein refers to an agent which helps to maintain enzyme stability during freeze-drying and storage by forming a protective glassy state and also helps increase the volume and mass of the lyophilized cake, improving its mechanical properties and reconstitution characteristics. For example, Trehalose, Sucrose, Mannitol, Sorbitol, Polyethylene glycol (PEG) and Hydroxypropyl methylcellulose (HPMC). Trehalose respectively have been found to be particularly suitable in the formulations described herein.

[0071] A "stable" formulation is one in which the enzyme therein substantially retains its physical stability and / or chemical stability and / or its biological activity upon storage. In one aspect, the formulation substantially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation.

[0072] A "sterile" formulation is aseptic or free from all living microorganisms and their spores.An "antioxidant" herein refers to an agent that inhibits the oxidation of other molecules. Examples of antioxidants herein include Methionine, citrate, lipoic acid, uric acid, glutathione, tocopherol, carotene, lycopene and cysteine.

[0073] A “free amino acids”, which act as stabilizers and enhancers of enzymatic activity. Examples of amino acids herein include Arginine, Serine, Tyrosine, Cysteine, Proline, Glutamine and Glycine.

[0074] A "cryoprotectant" herein refers to an agent that stabilizing enzyme structures during freeze-drying or storage. It prevents protein denaturation and maintains enzyme activity by reducing ice crystal formation, which can disrupt the enzyme's functional integrity. Examples of cryoprotectant herein include Glycerol, DMSO (Dimethyl sulfoxide), Polyethylene glycol (PEG) and Propylene glycol.

[0075] The present embodiment relates to an enzyme formulation may comprising “one or more salts”. Examples of salts herein include Sodium Chloride (NaCl), Magnesium Chloride (MgCE), Calcium Chloride (CaCE), Potassium Chloride (KC1), Ammonium Sulfate ((NH^SO^, Sodium Citrate and Sodium Phosphate.

[0076] The main embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) buffer; b) sugar / bulking agent; c) antioxidant; d) salt; and e) optionally, a free amino acid or cryoprotectant.

[0077] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; and e) optionally, Arginine or Glycerol.

[0078] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; e) Arginine; and f) Glycerol; wherein said formulation is surfactant-free.

[0079] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; and e) optionally, ImM to 60mM Arginine or 0.5% to 20% Glycerol; wherein said formulation has pH of about 6 to 8 and surfactant free.

[0080] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) about 10 mM Histidine buffer; c) about 119 mM Trehalose; d) about 10 mM Methionine; e) about 100 mM NaCl; and f) optionally, about 20 mM L-Arginine or about 10% Glycerol; wherein said formulation has pH of about 6.5 to 7.5.Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) 0.1 to 10 mg / mL Histidine buffer; c) 1 to 150 mg / mL Trehalose; d) 0.1 to 10 mg / mL Methionine; e) 1 to 20 mg / mL Sodium Chloride; and f) optionally, 0.174 to 10.452 mg / mL Arginine or 5 to 200 mg / mL Glycerol; wherein said formulation has pH of about 6 to 8.

[0081] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) ImM to 50mM Histidine buffer; c) 50mM to 160mM Trehalose; d) ImM to 50mM Methionine; e) 50mM to 150mM NaCl; f) ImM to 60mM Arginine; and g) 0.5% to 20% Glycerol; wherein said formulation has pH of about 6.5 to 7.5.

[0082] Another embodiment of the present invention is to provide a stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; wherein said formulation has pH of about 6 to 8 and surfactant free.

[0083] In another embodiment of the present invention is to provide a stable pharmaceutical composition of hyaluronidase enzyme, wherein composition is free of human serum albumin.

[0084] In another embodiment of the present invention is to provide a stable pharmaceutical composition of hyaluronidase enzyme, wherein composition is free of surfactant.

[0085] In another embodiment of the present invention, concentration of trehalose is of about 0-600 mM, 10-590 mM, 20-580 mM, 30-570 mM, 40-560 mM, 50-550 mM, 60-540mM, 70-530 mM, 80-520 mM, 90-510 mM, 100-500 mM, 110-490 mM, 120-480 mM, 130-470 mM, 140-460 mM, 150-450 mM, 160-440 mM, 170-430 mM or 200-300 mM More preferably the concentration of trehalose is of about 10-200 mM

[0086] In another embodiment of the present invention, concentration of NaCl is of about 0-600 mM, 10-590 mM, 20-580 mM, 30-570 mM, 40-560 mM, 50-550 mM, 60-540mM, 70-530 mM, 80-520 mM, 90-510 mM, 100-500 mM, 110-490 mM, 120-480 mM, 130-470 mM, 140-460 mM, 150-450 mM, 160-440 mM, 170-430 mM or 200-300 mM More preferably the concentration of NaCl is of about 10-200 mM.

[0087] In another embodiment of the present invention, concentration of histidine is of about 0-50 mM, 2-48 mM, 4-46 mM, 6-44 mM, 8-42 mM, 10- 40 mM, 12-38 mM, 14-36 mM, 16-34 mM, 18-32 mM, 20-30 mM, 22-28 mM, or 24-26 mM. More preferably the concentration of histidine is of about 1-50 mM.

[0088] In another embodiment of the present invention, concentration of methionine is of about 0-50 mM, 2- 48 mM, 4-46 mM, 6-44 mM, 8-42 mM, 10- 40 mM, 12-38 mM, 14-36 mM, 16-34 mM, 18-32 mM, 20-30 mM, 22-28 mM, or 24-26 mM. More preferably the concentration of methionine is of about 1-50 mM.

[0089] In another embodiment of the present invention, concentration of arginine or L-arginine is of about 1-100 mM, 15-95 mM, 20-90 mM, 25-85 mM, 30-80 mM, 35-75 mM, 40-70 mM, 45-65 mM, 50-60 mM, or 55-65 mM. More preferably the concentration of arginine or L-arginine is of about 1-70 mM.

[0090] In another embodiment of the present invention, concentration of glycerol is of about 0.1-100%, 1-90%, 2-80%, 3-70%, 4-60%, 5-50%, 6-40% or 7-30%, More preferably the concentration of glycerol is of about 0.1-20%.

[0091] The detailed description provided is exemplary in nature and is not intended to limit the application or uses of the invention. The following examples further illustrate the invention, but are not meant to limit its scope. Various changes and modifications may be made by those skilled in the art based on the description of the invention, and such changes and modifications are also encompassed within the scope of the present invention.

[0092] EXAMPLES

[0093] EXPERIMENTAL DETAILS:

[0094] Temperatures evaluated:

[0095] The stability of hyaluronidase was assessed under the following temperature conditions:

[0096] 1. Room Temperature (22-28°C): This range simulates typical storage conditions but is expected to lead to gradual enzyme degradation over time.

[0097] 2. Refrigeration (2-8°C): This temperature range slows down enzyme activity loss but may not fully prevent degradation without stabilizers.

[0098] 3. Deep freezing (-80°C): The optimal storage condition for long-term enzyme preservation, with minimal degradation.

[0099]

[0100]

[0101] Table 1: Evaluated four different types of formulation buffer to stabilize of drug substance

[0102] EXAMPLE 1: STABILITY STUDY ANALYSIS

[0103] Purity level of formulation Fl by RP-HPLC

[0104] Formulation Fl was kept at different storage condition of 2-8°C and -80°C temperature for 90 Days and evaluated by RP-HPLC analysis. The results showed on Day 0 purity greater than 92%, on Day 7 & Day 30 purity remains greater than 92%, and on Day 60 & Day 90 at 2-8°C purity greater than 92%. (Refer figure 1 to figure 7).

[0105] Purity level of formulation F2 by RP-HPLC

[0106] Formulation F2 was kept at different storage condition of 2-8°C and -80°C temperature for 90 Days and evaluated by RP-HPLC analysis. The results showed on Day 7 & Day 30 purity greater than 92%, and on Day 60 & Day 90 at 2-8°C purity remained greater than 92%. (Refer figure 8, 9, 19 & 20).

[0107] Purity level of formulation F3 by RP-HPLC

[0108] Formulation F3 was kept at different storage condition of 2-8°C and -80°C temperature for 90 Days and evaluated by RP-HPLC analysis. The results showed on Day 7 & Day 30 purity greater than 92%, and on Day 60 & Day 90 at 2-8°C purity remained greater than 92%. (Refer figure 10, 11, 17 & 18).

[0109] Purity level of formulation F4 by RP-HPLC

[0110] Formulation F4 was kept at different storage condition of 2-8°C and -80°C temperature for 90 Days and evaluated by RP-HPLC analysis. The results showed on Day 7 & Day 30 purity greater than 92%, and on Day 90 at 2-8°C purity remained greater than 92%. (Refer figure 12, 21 & 22).

[0111] EXAMPLE 2: ENZYME STABILITY EVALUATION

[0112] The enzyme stability was evaluated over a period of up to three months, and different temperature conditions were tested to assess the impact of formulation buffers on enzyme activity. Measurements were taken at various time points to determine the optimal formulation and storage conditions for maintaining hyaluronidase activity.To achieve maximum stability for hyaluronidase, it is essential to use a carefully optimized buffer composition with appropriate stabilizers. Among the formulation strategies tested, the combination of trehalose, methionine, L-arginine, and glycerol offers the best protection against temperature fluctuations, oxidative damage, aggregation, and freezing-induced denaturation. The use of these stabilizing agents ensures that the enzyme remains active and functional, even after extended storage under challenging conditions.

[0113] The below data, up to 10 days hyaluronidase is stable with > 96% purity and >90000 enzyme activity / mL at room temperature (22-28°C).

[0114]

[0115] Table 2: Stability study data at RT for 10 days

[0116]

[0117] Table 3: Hyaluronidase Bioactivity

[0118]

[0119] Table 4: Stability study data at 2-8°C, -80°CComparative analysis was performed between Fl, F2, F3 & F4 at -80°C & 2-8°C for 0, 7, & 30 days (Refer figure 15 to 22). Comparative trends shows that all the formulation are comparable from Day 0 to Day 30.

[0120]

[0121] Table 5: Representative profile of RP HPLC purified Hyaluronidase Based on the above study, Hyaluronidase enzyme is very much stable up to 3 months at 2-8°C with >92% purity.

[0122] Three batches of Hyaluronidase were formulated in the formulation strategy 1 (Fl) and referred as Batch 1, Batch 2 and Batch 3 here after. These batches were charged on (-20°C) and (2°C-8°C). EXAMPLE 3: Stability data of Hyaluronidase Drug Substance for 12M AT (-20°C)

[0123] A) Physical appearance

[0124] Over a 12 month evaluation period for RT condition (-20°C), the samples from all three tested batches were consistently maintained within the specified compliance range.

[0125] B) pH

[0126]

[0127] Table 6: Results of pH of all three batches for 12M at -20°C Observation:

[0128] Based on the 12 months data, the pH of all the 3 batches were found to be comparable and remained within the specified compliance range, showing only minor fluctuations as depicted in Figure 25.C) OSMOLALITY

[0129]

[0130] Table 7: Results of Osmolality of all three batches for 12M at -20°C D) RP - HPLC:

[0131]

[0132] Table 8: Result of RP -HPLC of all three batches for 12M at -20°CObservation:

[0133] Based on 12M RP-HPLC data, the protein concentration of three hyaluronidase batches formulated in Fl are remained stable as depicted in Figure 27-29.

[0134] E) ENZYME ACTIVITY:

[0135]

[0136] Table 9: Result of enzymatic activity of all three batches for 12M at -20°C Observation:

[0137] Based on 12M enzyme activity data, the enzyme activity of all three hyaluronidase batches formulation in Fl remain stable with minor fluctuation as depicted in Figure 30.

[0138] D) SE- HPLC:

[0139]

[0140]

[0141] Table 10: Data of SE-HPLC of all three batches for 12M at -20°C Observation:

[0142] Based on 12 moths -20°C data, the qualitative and quantitative attributes of the three Hyaluronidase batches formulated in Formulation buffer 1 are found to be stable.

[0143] EXAMPLE 4: Stability data of Hyaluronidase Drug Substance for 12M AT (2-8°C)

[0144] A) PHYSICAL APPEARANCE

[0145] Over a 12-month evaluation period for AT condition (2-8°C), the samples from all three tested batches were consistently maintained within the specified compliance range.

[0146] B) pH

[0147]

[0148] Table 11: Results of pH of all three batches for 12M at 2-8°C Observation:

[0149] Based on the 12 months data, the pH of all the 3 batches were found to be comparable and remained within the specified compliance range, showing only minor fluctuations as depicted in Figure 33. C) OSMOLALITY

[0150]

[0151] Table 12: Results of Osmolality of all three batches for 12M at 2-8°CD) RP - HPLC:

[0152]

[0153] Table 13: Data of RP-HPLC of all three batches for 12M at 2-8°C Observation:

[0154] Based on 12M RP-HPLC data, the protein concentration of three hyaluronidase batches formulated in Fl are remained stable as depicted in Figure 35-37.E) ENZYME ACTIVITY:

[0155]

[0156] Table 14: Results of enzymatic activity of all three batches for 12M at 2 - 8°C Observation:

[0157] Based on 12M enzyme activity data, the enzyme activity of all three hyaluronidase batches formulation in Fl remain stable with minor fluctuation as depicted in Figure 38.

[0158] D) SE- HPLC:

[0159]

[0160] Table 15: Data of SE-HPLC of all three batches for 12M at 2-8°CObservation:

[0161] Based on 12 moths 2-8°C data, the qualitative and quantitative attributes of the three Hyaluronidase batches formulated in Formulation buffer 1 are found to be stable.

Claims

We claim,1. A stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) buffer; b) sugar / bulking agent; c) antioxidant; d) salt; and e) optionally, a free amino acid or cryoprotectant.

2. The pharmaceutical composition of claim 1 , wherein the composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; and e) optionally, Arginine or Glycerol.

3. The pharmaceutical composition of claim 1 , wherein the composition of a hyaluronidase enzyme comprising: a) Histidine buffer; b) Trehalose; c) Methionine; d) NaCl; e) Arginine; and f) Glycerol; wherein said formulation is surfactant-free.

4. A stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; and e) optionally, ImM to 60mM Arginine or 0.5% to 20% Glycerol; wherein said formulation has pH of about 6 to 8 and surfactant free.

5. The pharmaceutical composition of claim 4, wherein the composition of hyaluronidase enzyme comprising: a) 0.5 to 2 mg / mL with 90000 to 120000 lU / mL activity Hyaluronidase; b) 0.1 to 10 mg / mL Histidine buffer; c) 1 to 150 mg / mL Trehalose; d) 0.1 to 10 mg / mL Methionine; e) 1 to 20 mg / mL Sodium Chloride; and f) optionally, 0.174 to 10.452 mg / mL Arginine or 5 to 200 mg / mL Glycerol; wherein said formulation has pH of about 6 to 8.

6. A stable pharmaceutical composition of a hyaluronidase enzyme comprising: a) ImM to 50mM Histidine buffer; b) 50mM to 160mM Trehalose; c) 1 mM to 50mM Methionine; d) 50mM to 150mM NaCl; wherein said formulation has pH of about 6 to 8 and surfactant free.

7. The pharmaceutical composition of claim 6, wherein the composition of hyaluronidase enzyme is free of surfactant.

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