Method for purification of hyaluronidase enzyme

Abstract

The present invention relates to method for purification of hyaluronidase enzyme comprising three chromatographic purification methods for obtaining purified hyaluronidase enzyme, wherein first Anion exchange chromatography, second is pseudo affinity chromatography followed by multimodal chromatography.

Description

[0001] METHOD FOR PURIFICATION OF HYALURONIDASE ENZYME

[0002] RELATED APPLICATIONS

[0003] This application is related to Indian Provisional Application IN202421053456 filed 12thJuly, 2024 and is incorporated herein in its entirely.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a method for purification of human recombinant hyaluronidase enzyme.

[0006] BACKGROUND OF THE INVENTION

[0007] 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 catalyzing 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, drugs, 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 methods for the production and purification of hyaluronidase.

[0008] Recombinant human hyaluronidase (rHuPH20) is a glycosylated single chain protein with 447 amino acids, with calculated molecular weight of full-length polypeptide is 51106 Dalton, and with glycosylated, molecular weight is 60000-65000 Da. There are six N-linked (47, 131, 200, 219, 333, 358) and one O- linked glycosylation site and 12 cysteines with 6 Di-sulfide linkages which makes it a complex during purification.

[0009] WO2014203133 discloses a purification method for bacterial Hyaluronidase by using ion-exchange chromatography and weak cation-exchange chromatography.

[0010] W02004092361 discloses a purification method for mammalian Hyaluronidase by using viral filtration method for increased purity of Hyaluronidase.

[0011] Thus, there is a need in the art for identification of methods that can be used for purification of human recombinant hyaluronidase to increase the purity of the final product. The present invention addresses this critical issue by proposing a three-step chromatography purification method for purification of human recombinant hyaluronidase.

[0012] OBJECTS OF THE INVENTION The main object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises pseudo affinity chromatography and multimodal chromatography.

[0013] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises only two chromatographic steps first pseudo affinity chromatography followed by in flow through mode with second multi modal affinity chromatography.

[0014] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises anion exchange chromatography, pseudo affinity chromatography and multimodal chromatography.

[0015] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises three chromatographic steps first anion exchange chromatography followed by second pseudo affinity chromatography and third multimodal chromatography.

[0016] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein anion exchange chromatography comprises following buffer composition: a) Equilibration with equilibration buffer: 20 mM Sodium Phosphate; 5 mM EDTA; lOmM Methionine; 119 mM Trehalose dehydrate; and pH 7.5; and b) Elution by elution buffer: 20 mM Sodium Phosphate; 0.4 M NaCl; 5 mM EDTA; 10 mM L- Methionine; 119 mM Trehalose dehydrate; and pH 7.5.

[0017] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein pseudo affinity chromatography comprises following buffer composition:

[0018] • Equilibration with equilibration buffer: a) lOmM sodium phosphate, 5mM EDTA, pH 6.0; or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

[0019] • Elution by elution buffer: a) lOmM sodium phosphate, 0.5M KC1, 5mM EDTA, pH 6.0 or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 640 mM NaCl, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

[0020] Another object of the present invention is to provide a method for purification of Hyaluronidase enzyme, wherein multimodal chromatography comprises following buffer composition:

[0021] • Equilibration with equilibration buffer: a) 20mM Tris, lOOmM KC1, and pH 8.0 or b) 20 mM Tris, lOOmM NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0022] • Elution by elution buffer: a) 20mM Tris, IM KC1, pH 8.0 or b) 20 mM Tris, 1.5 M NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0023] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein multimodal chromatography comprises two functionalities first anion exchange and second hydrophobic interaction.

[0024] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest Clarification; b) Ultrafiltration and Diafiltration I; c) Pseudo Affinity Chromatography; d) Multimodal Chromatography; e) Ultrafiltration and Diafiltration II; and f) 0.22 pm filtration.

[0025] Another object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest clarification; b) Tangential flow filtration I; c) Anion exchange chromatography; d) Tangential flow filtration II; e) Pseudo affinity chromatography; f) Multimodal chromatography; and g) Tangential flow filtration III and DS preparation.

[0026] SUMMARY OF THE INVENTION

[0027] The aspect object of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises pseudo affinity chromatography and multimodal chromatography. Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises only two chromatographic steps first pseudo affinity chromatography followed by in flow through mode with second multi modal affinity chromatography.

[0028] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises anion exchange chromatography, pseudo affinity chromatography and multimodal chromatography.

[0029] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises three chromatographic steps first anion exchange chromatography followed by second pseudo affinity chromatography and third multimodal chromatography.

[0030] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein anion exchange chromatography comprises following buffer composition: a) Equilibration with equilibration buffer: 20 mM Sodium Phosphate; 5 mM EDTA; lOmM Methionine; 119 mM Trehalose dehydrate; and pH 7.5; and b) Elution by elution buffer: 20 mM Sodium Phosphate; 0.4 M NaCl; 5 mM EDTA; 10 mM L- Methionine; 119 mM Trehalose dehydrate; and pH 7.5.

[0031] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein pseudo affinity chromatography comprises following buffer composition:

[0032] • Equilibration with equilibration buffer: a) lOmM sodium phosphate, 5mM EDTA, pH 6.0; or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

[0033] • Elution by elution buffer: a) lOmM sodium phosphate, 0.5M KC1, 5mM EDTA, pH 6.0 or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 640 mM NaCl, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

[0034] Another aspect of the present invention is to provide a method for purification of Hyaluronidase enzyme; wherein multimodal chromatography comprises following buffer composition:

[0035] • Equilibration with equilibration buffer: a) 20mM Tris, lOOmM KC1, and pH 8.0 or b) 20 mM Tris, lOOmM NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0036] • Elution by elution buffer: a) 20mM Tris, IM KC1, pH 8.0 or b) 20 mM Tris, 1.5 M NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0037] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein multimodal chromatography comprises two functionalities first anion exchange and second hydrophobic interaction.

[0038] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest Clarification; b) Ultrafiltration and Diafiltration I; c) Pseudo Affinity Chromatography; d) Multimodal Chromatography; e) Ultrafiltration and Diafiltration II; and f) 0.22 pm filtration.

[0039] Another aspect of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest clarification; b) Tangential flow filtration I; c) Anion exchange chromatography; d) Tangential flow filtration II; e) Pseudo affinity chromatography; f) Multimodal chromatography; and g) Tangential flow filtration III and DS preparation.

[0040] BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 : Representative chromatogram of Pseudo Affinity Chromatography

[0042] Figure 2: Representative chromatogram of MMC Chromatography

[0043] Figure 3: Representative SDS-PAGE profile

[0044] Figure 4: Representative profile of RP-HPLC purified Hyaluronidase

[0045] Figure 5: Representative profile of SE-HPLC purified Hyaluronidase

[0046] Figure 6: SDS-PAGE (NR) for three batch drug substance Figure 7: SDS-PAGE (R) for three batch drug substance

[0047] Figure 8: Three consistency batches Quantification and Purity - RP-HPLC

[0048] Figure 9: Three consistency batches Purity - SEC HPLC

[0049] Figure 10: Representative chromatogram of anion exchange Chromatography

[0050] DETAILED DESCRIPTION OF THE INVENTION

[0051] The following is a detailed description of embodiments of the invention. The embodiments are in such details as clearly communicate the invention. However, the number 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.

[0052] DEFINITION

[0053] The following definitions are provided to facilitate understanding of certain terms used throughout the specification.

[0054] 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.

[0055] 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.

[0056] 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. 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.

[0057] 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.

[0058] The “Ultrafiltration”, “Diafiltration”, form a comprehensive membrane-based filtration system for separating particles and molecules across different size ranges. Ultrafiltration employs membranes with pore sizes typically ranging from 1 to 100 manometer, effectively removing macromolecules and colloids. Diafiltration extends this process by continuously replacing permeate with fresh solvent to enhance purification and concentration.

[0059] The “pseudo-affinity chromatography” employs dyes as ligands to bind proteins. These dyes mimic natural ligands but lack high specificity, allowing them to capture a diverse range of proteins.

[0060] The “multimodal chromatography” utilizes media supports functionalized with ligands capable of multiple interaction modes: ion exchange, hydroxyapatite, affinity, size exclusion, and hydrophobic interactions.

[0061] The main embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises pseudo affinity chromatography and multimodal chromatography.

[0062] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises only two chromatographic steps first pseudo affinity chromatography followed by in flow through mode with second multi modal affinity chromatography.

[0063] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises anion exchange chromatography, pseudo affinity chromatography and multimodal chromatography.

[0064] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises three chromatographic steps first anion exchange chromatography followed by second pseudo affinity chromatography and third multimodal chromatography.

[0065] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein anion exchange chromatography comprises following buffer composition:

[0066] • Equilibration with equilibration buffer: 20 mM Sodium Phosphate; 5 mM EDTA; lOmM Methionine; 119 mM Trehalose dehydrate; and pH 7.5; and

[0067] • Elution by elution buffer: 20 mM Sodium Phosphate; 0.4 M NaCl; 5 mM EDTA; 10 mM L- Methionine; 119 mM Trehalose dehydrate; and pH 7.5.

[0068] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein pseudo affinity chromatography comprises following buffer composition:

[0069] • Equilibration with equilibration buffer: a) lOmM sodium phosphate, 5mM EDTA, pH 6.0; or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0. • Elution by elution buffer: a) lOmM sodium phosphate, 0.5M KC1, 5mM EDTA, pH 6.0 or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 640 mM NaCl, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

[0070] Another embodiment of the present invention is to provide a method for purification of Hyaluronidase enzyme, wherein multimodal chromatography comprises following buffer composition:

[0071] • Equilibration with equilibration buffer: a) 20mM Tris, lOOmM KC1, and pH 8.0 or b) 20 mM Tris, lOOmM NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0072] • Elution by elution buffer: a) 20mM Tris, IM KC1, pH 8.0 or b) 20 mM Tris, 1.5 M NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

[0073] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein multimodal chromatography comprises two functionalities first anion exchange and second hydrophobic interaction.

[0074] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest Clarification; b) Ultrafiltration and Diafiltration I; c) Pseudo Affinity Chromatography; d) Multimodal Chromatography; e) Ultrafiltration and Diafiltration II; and f) 0.22 pm filtration.

[0075] Another embodiment of the present invention is to provide a method for purification of hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest clarification; b) Tangential flow filtration I; c) Anion exchange chromatography; d) Tangential flow filtration II; e) Pseudo affinity chromatography; f) Multimodal chromatography; and g) Tangential flow filtration III and DS preparation.

[0076] In another embodiment of the present invention a method for purification of hyaluronidase enzyme, wherein the cell culture harvest after 0.22micron filtration processed through tangential flow filtration I comprises TFF I buffer: lOmM sodium phosphate; 5mM EDTA; and pH 6.0.

[0077] In another embodiment of the present invention a method for purification of hyaluronidase enzyme, wherein tangential flow filtration III processed after multimodal chromatography. Wherein TFF III comprises TFF III buffer: 1 mM L-Histidine HC1, 8 mM L-Histidine, 1 mM EDTA, 10 mM L-Methionine, 119 mM Trehalose dihydrate, 100 mM NaCl, pH 7.0.

[0078] In another embodiment of the present invention a method for purification of hyaluronidase enzyme, wherein multimodal chromatography output is processed through tangential flow filtration II comprises TFF II buffer: 20mM histidine-HCl, pH 7.2.

[0079] In another embodiment of the present invention, the purified Hyaluronidase enzyme can be used in combination with antibody.

[0080] In another embodiment of the present invention it provides a three-step chromatographic purification method for obtaining high-purity hyaluronidase having more than 99 % purity and more than ~ 90,000 lU / mg in activity.

[0081] In another embodiment of the present invention the purified Hyaluronidase enzyme can be used in combination with Trastuzumab, Nivolumab, Golimumab, Pembrolizumab, Denosumab, Bevacizumab, Adalimumab, Rituximab, Ustekinumab, Daratumumab, Dupilumab or Phesgo.

[0082] The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also from the part of this specification and are intended to be included within the scope of it.

[0083] EXPERIMENTAL DETAILS:

[0084] EXAMPLE 1: PURIFICATION OF HYALURONIDASE ENZYME USING PSEUDO AFFINITY AND MULTIMODAL CHROMATOGRAPHY

[0085] The cells were harvested from the bioreactor and processed through a cell removal and clarification system to separate the cell culture fluid containing the hyaluronidase from cells and cell debris. In addition to concentrating the protein, the cell culture media has been exchanged with an appropriate buffer at this time while addition of the chelating agent i.e. EDTA. Following the addition of EDTA, the cell culture harvest after 0.22micron filtration, processed through tangential flow filtration (TFF I Buffer: lOmM Sodium Phosphate + 5mM EDTA, pH 6.0) to remove media components and exchange it into the buffer matrix for the next step - pseudo affinity chromatography.

[0086] Concentrated and buffer exchanged cell culture supernatant was then processed through pseudo affinity chromatography. Affinity of the resin to protein of interest plays an important role in the purification of the hyaluronidase by pseudo affinity chromatography. The binding of the protein of interest mainly exploits the glycosylation sites which are present on the protein. As hyaluronidase has 6 N linked and one O-linked glycosylation sites it was prone to show affinity towards Cellufine MAX DexS-Hb.

[0087] Pseudo affinity chromatography was operated in bind and elute mode (Capture Equilibration Buffer: lOmM Sodium Phosphate + 5mM EDTA, pH 6.0) wherein lower conductivity (less 2 mS / cm) at pH 6 facilitates the binding of the protein of interest to the column.

[0088] Elution (Capture Elution Buffer: lOmM Sodium Phosphate + 0.5M KC1 + 5mM EDTA, pH 6.0) was performed by increasing the salt concentration of the buffer over a step gradient of 25%. The impurity fraction was eluted earlier at 8% of step elution and protein of interest elutes at 25% gradient.

[0089] As the input was loaded on to the column, packed with resin, the hyaluronidase enzyme was observed to bind to the resin while the media components along with the impurities were washed in flow through. Following loading of the input, a post load wash was given with the equilibration buffer and collected with the product. Post collection of the desired product at 25% gradient with peak-to-peak collection, impurities bound to the resin were stripped off by passing 100% gradient followed by water through the column and collected separately, thereby regenerating the resin.

[0090] The Affinity output was conditioned by adding WFI to adjust conductivity lesser than 10 mS / cm and pH 8.0 with the help of tris base, which now acts as a Mixed Mode Chromatography (Capto adhere) resin input.

[0091] Multimodal chromatography (MMC equilibration buffer: 20mM Tris + lOOmM KC1, pH 8.0) was operated in bind and elute mode with the two functionalities i.e. AEX and HIC where in lower conductivity (less than 10 mS / cm) at pH 8.0, facilitating the binding, protein of interest to the column.

[0092] Elution (MMC Elution Buffer: 20mM Tris + IM KC1, pH 8.0) was performed by addition of the salt concentration of the buffer over a linear gradient wherein protein of interest elutes up to 50% gradient.

[0093] As the input was loaded on to the column, packed with resin, the hyaluronidase enzyme along with the impurities were observed to bind the resin, while the unbound impurities flow through the column and are collected. Following loading of the input, a post load wash was given with the equilibration buffer and collected with the product. Post collection of the desired product at 35-45% gradient with peak-to-peak collection, impurities bound to the resin were stripped off by passing 100% gradient followed by water through the column and collected separately, thereby regenerating the resin. MMC output processed through TFF-II (TFF-II Buffer: 20mM Histidine-HCl, pH 7.0-7.2) to concentrate (> Img / mL) and buffer exchange the product. The bioactivity was 43000 to 99600 unit / mg protein concentration (Table 2).

[0094] Table 1. Downstream process flow

[0095] Table 2: Hyaluronidase Bioactivity

[0096] EXAMPLE 2: PURIFICATION OF HYALURONIDASE ENZYME USING ANION EXCHANGE, PSEUDO AFFINITY AND MULTIMODAL CHROMATOGRAPHY

[0097] Table 3: Downstream process flow

[0098] Hyaluronidase was purified using three chromatography as described in table 3 and three batches were prepared for further analysis. Following table 4 describes the quality data for all three batches.

[0099] Table 4: Quality data

[0100] All three-chromatography output of the present invention was analysed for step yield (%), host cell DNA content (ng / mL), and enzyme activity (U / rnL) for all three batches (Table 5-7).

[0101] Table 5: Step yield in all three batches

[0102] Table 6: Host Cell DNA in all three batches

[0103] Table 7: Enzyme activity in all three batches

[0104] Final drug substance quality was checked by following methods for desired parameters and analysed quality attributed based on the acceptance criteria (Table 8).

[0105] Table 8: Drug substance quality

[0106] Table 9 represents the purity and concentration of hyaluronidase in all three batches measured by reverse phase HPLC; Table 10 describes the total percentage of impurities found using SEC-HPLC method; and Table 11 describes the enzyme activity, host cell protein and host cell DNA data for all three consistency batches. Table 9: Three Consistency batches concentration and purity by RP-HPLC

[0107] Table 10: Consistency batches Purity - SEC HPLC

[0108] Table 11: Three Consistency batches Enzyme activity, HCP and HCDNA data.

[0109] Above-described method of the present invention for the purification of enzyme purify the hyaluronidase without affecting the enzyme activity. Hence, above mention method suits for the purification of hyaluronidase enzyme.

Claims

We claim,1. A method for purification of a hyaluronidase enzyme, wherein the method comprises pseudo affinity chromatography and multimodal chromatography.

2. A method for purification of a hyaluronidase enzyme, wherein the method comprises anion exchange chromatography, pseudo affinity chromatography and multimodal chromatography.

3. The method according to claim 2, wherein the method comprises three chromatographic steps first anion exchange chromatography followed by second pseudo affinity chromatography and third multimodal chromatography.

4. The method for the purification of hyaluronidase enzyme according to any of the preceding claim, wherein multimodal chromatography comprises two functionalities first anion exchange and second hydrophobic interaction.

5. A method for purification of Hyaluronidase enzyme, wherein the method comprises steps of: a) Harvest clarification; b) Tangential flow filtration I; c) Anion exchange chromatography; d) Tangential flow filtration II; e) Pseudo affinity chromatography; f) Multimodal chromatography; and g) Tangential flow filtration III and DS preparation.

6. The method for the purification of hyaluronidase according to any of preceding claim, wherein anion exchange chromatography comprises following buffer composition: a) Equilibration with equilibration buffer: 20 mM Sodium Phosphate; 5 mM EDTA; 10 mM Methionine; 119 mM Trehalose dehydrate; and pH 7.5; and b) Elution by elution buffer: 20 mM Sodium Phosphate; 0.4 NaCl; 5 mM EDTA; 10 mM L- Methionine; 119 mM Trehalose dehydrate; and pH 7.5.

7. The method for the purification of hyaluronidase according to any of preceding claim, wherein pseudo affinity chromatography comprises following buffer composition: i) Equilibration with equilibration buffer: a) lOmM sodium phosphate, 5mM EDTA, pH 6.0; or b) 10 mM Sodium Phosphate, 10 mM L-Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.

0. ii) Elution by elution buffer: a) lOmM sodium phosphate, 0.5M KC1, 5mM EDTA, pH 6.0; orb) 10 mM Sodium Phosphate, 10 mM L-Methionine, 640 mM NaCl, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 7.0.

8. The method for the purification of hyaluronidase according to any of preceding claim, wherein multimodal chromatography comprises following buffer composition: i) Equilibration with equilibration buffer: a) 20mM Tris, lOOmM KC1, and pH 8.0 or b) 20 mM Tris, lOOmM NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.

0. ii) Elution by elution buffer: a) 20mM Tris, IM KC1, pH 8.0; or b) 20 mM Tris, 1.5 M NaCl, 10 mM L- Methionine, 119 mM Trehalose dihydrate, 5 mM EDTA, pH 8.0.

9. The purified hyaluronidase enzyme according to any of the preceding claim, wherein hyaluronidase can be used on combination with antibody.

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