KR102350592B1 - Purification method of hyaluronidase - Google Patents

Abstract

The present invention relates to a method for refining hyaluronidase which comprises: a step of performing affinity chromatography; a step of performing cation exchange chromatography using sulfopropyl sepharose; and a step of performing size exclusion chromatography. According to the present invention, high purity of hyaluronidase can be cost effectively obtained.

Description

Purification method of hyaluronidase

The present invention relates to a method for purifying hyaluronidase.

Hyaluronidase (hyaluronidase, HAdase) is a generic term for enzymes that reduce the molecular weight of hyaluronic acid (HA). Depending on the mechanism of hydrolysis of hyaluronic acid, mammalian type hyaluronidase (Mammalian type, EC 3.2.1.35, hyaluronoglucosaminidase), leech type hyaluronidase (Leeches type, EC 3.2.1.36, hyaluronoglucuronidase) and bacteria It is classified as a type 　 hyaluronidase 　 (Bacterial type, EC 4.2.2.1, hyaluronate lyase).

In particular, mammalian-type hyaluronidase exists in the testes, skin, liver, and placental body fluids in the human body and hydrolyzes the β-1, tetrasaccharide bond between glucuronic acid and glucosamine, which are components of hyaluronic acid, to form tetrasaccharides (tetrasaccharides). ), or hydrolyzes chondroitin, chondroitin-4-sulfuric acid, and chondroitin-6-sulfuric acid, which are components of synovial fluid and cartilage in our joints.

Hyaluronidase is used for infiltration and blocking anesthesia to increase the diffusion of local anesthetics and steroids in orthopedic, ophthalmic, plastic surgery, dental, oral surgery, gynecological and otolaryngeal surgery, dispersing body fluids such as hematomas, It is used to prevent peritoneal adhesions, prevent stone formation, and treat infertility.

The conventional hyaluronidase purification method was complicated and produced by many processes, so it was difficult to use commercially (KR 10-2002-0011138 A). In addition, the hyaluronidase obtained by the conventional method has many problems such as animal-derived allergy, anaphylactic reaction, mad cow disease infection, etc. due to its low purity.

Therefore, there is a need for an improved purification method for purifying hyaluronidase with high purity in an economical and simple manner.

It is an object of the present invention to provide a method for purifying hyaluronidase.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

One aspect of the present invention provides a method for purifying hyaluronidase (HAdase).

Specifically, the purification method comprises the steps of: (a) performing affinity chromatography (AC) on a hyaluronidase-containing raw material; (b) performing cation exchange chromatography (CEX); And (c) it may be a purification method proceeding in the order of performing size exclusion chromatography (SEC).

As used herein, the term “the hyaluronidase-containing raw material” may be an unrefined raw material or a partially purified hyaluronidase-containing raw material.

The term "hyaluronidinase" is a generic term for enzymes that lower molecular weight hyaluronic acid (HA). By catalyzing the hydrolysis of hyaluronic acid, hyaluronidase lowers the viscosity of hyaluronic acid, thereby improving tissue permeability. increase The hyaluronidase is usually derived from mammalian animals, for example, enzymes derived from testes of humans, cattle, sheep, pigs, etc., or recombinantly expressed by introducing mammalian-derived hyaluronidase into microorganisms, animal cells, or plant cells. It may be hyaluronidase. The production method of recombinant hyaluronidase is the same as that of production in microorganisms, animal cells, or plant cells according to the conventional recombinant method of mammalian genes (Frost et al., 1997, BBRC, 236, 10-15; Lin et al. al., 1993, Proc. Natl. Acad. Sci. USA, 90, 10071-10075; Reitinger et al., 2008, Protein Expression and Purification, 57, 226-233; Kordowicz et al., European patent, WO2000/077221 ).

The hyaluronidase according to the present invention may be an enzyme derived from a mammal, for example, a testis of a human, cow, sheep, or pig. A commercially available hyaluronidase is a form obtained by salting out mammalian testicles and then freeze-drying, or salting out, removing pyrogens and freeze-drying. For example, raw materials that are lyophilized after salting out once in mammalian testicles, raw materials that are freeze-dried after salting out twice in testicles, and freeze-dried after salting out twice in testicles to remove pyrogens, etc. can be used as raw materials in the purification process. can Conventional hyaluronidase is salted out twice in ovine testicles, and then undergoes lyophilization, dialysis, depyrogenation, and freeze-drying processes. It is extracted, and a large amount of protein is mixed with the extracted thing in addition to hyaluronidase. Accordingly, a purification process for removing impurities is required.

In addition, hyaluronidase is used, for example, as a spreading or dispersing agent with other agents, drugs and proteins to enhance its dispersibility and deliverability. Hyaluronidase is also used as other therapeutic agents and cosmetic ingredients. Due to the recent increase in the use of hyaluronidase, a large amount of purified hyaluronidase is required.

To this end, in the present invention, by effectively arranging the purification process sequence, the purification yield of hyaluronidase is improved, and by maximizing the removal of impurities through optimization of process steps, the number of purification processes is simplified and the efficiency of process operation is increased.

As used herein, the term “chromatography” refers to any process in which the components of a mixture are separated by passing a mixture through a medium such that the components of the mixture pass through the medium at different rates.

Chromatography of the present invention includes, but is not limited to, column chromatography, planar chromatography, thin layer chromatography, gas chromatography, liquid chromatography, rapid protein liquid chromatography (FPLC), and high performance liquid chromatography (HPLC). doesn't happen An exemplary type of chromatography process or apparatus in each step of the purification process of the present invention is disclosed, which is applicable to all types of chromatography described above.

The purification method of the present invention may be one in which steps (a) to (c) are performed only once and additionally the same or different chromatography is not performed.

The other chromatography may be at least one selected from the group consisting of dye affinity chromatography, reverse phase chromatography, and anion exchange chromatography, but is not limited thereto. does not

The purification method of the present invention is significant in that the purification yield can be improved by removing impurities of hyaluronidinase even when steps (a) to (c) are performed only once.

Each step of the method for purifying the hyaluronidase will be described in detail as follows. First, step (a) is a step of performing affinity chromatography.

Affinity chromatography used in the present invention prepares an antibody against a physiologically active substance to be purified, covalently binds it to a solid carrier, and then adds a sample containing the physiologically active substance to be purified to the antibody-carrier complex to produce a physiologically active substance It refers to a method of eluting and obtaining a target substance by adsorbing to the antibody, washing with an appropriate method, and then adding an eluent.

In one embodiment, the functional group of the resin of the affinity chromatography is protein A (protein A), heparin, blue (blue), benzamidine (benzamidine), metal ions (cobalt, nickel, copper) and hyaluronic acid. It may be any one selected from the group consisting of antibodies to Ronidase, but is not limited thereto.

For example, the column that can be used for affinity chromatography of the present invention is Blue Sepharose, Blue Sepharose Fast Flow (FF), Heparin Sepharose HP (Heparin Sepharose High Performance), or Heparin Sepharose FF. (Heparin Sepharose Fast Flow) may be used, and specifically, Blue Sepharose may be used, but it is not limited as long as it can isolate the desired hyaluronidase.

For example, a glycine buffer may be used as the column buffer for the affinity chromatography. For example, the concentration of the column buffer may be adjusted to about 1 mM to 100 mM, for example, 5 mM to 50 mM, or 10 mM to 30 mM. For example, as the buffer, a buffer of pH 8 to 12 may be used. In one example, the flow rate may be from about 1.0 ml/min to 20.0 ml/min or from about 5 ml/min to 10 ml/min. However, it is not limited thereto.

For the purpose of the present invention, affinity chromatography may be to perform any one or more steps of a sample injection step, an equilibration step, a washing step, and an elution step.

The chromatography of the present invention may be characterized in that it does not contain an additional stabilizer other than the buffer.

The additional stabilizer may include, but is not limited to, nonionic surfactants, chelating agents, or chlorides of alkali metals and alkaline earth metals.

The nonionic surfactant may be a polyoxyethylene sorbitan fatty acid ester (Tween). For example, it may be selected from the group consisting of polysorbate 20 (Tween20), polysorbate 80 (Tween80), and Triton X-100 (Triton X-100).

In addition, the chelating agent refers to a molecule including two or more electron-donating atoms capable of forming a coordination bond with a single metal ion, and may specifically be ethylenediaminetetraacetic acid (EDTA). The alkali metal and alkaline earth metal chloride may be _{magnesium chloride (MgCl 2 ).}

In any one of the above embodiments, before step (a) of the purification method of the present invention, the step of equilibrating the column using a buffer may be further included.

In the present invention, equilibration may refer to a step of stabilizing the column with a buffer solution in order to prevent protein aggregation or loss of activity due to changes in the environment before the protein to be purified is injected into the column.

Conditions such as flow rate, temperature, time and conductivity for flowing the buffer in the equilibration step before step (a) may be appropriately adjusted.

For example, a glycine buffer may be used as the buffer, and the flow rate may be about 1.0 ml/min to 20.0 ml/min or 5.0 ml/min to 10.0 ml/min. For example, the conductivity may be about 0 to 2 mS/cm. However, it is not limited thereto.

In the present invention, the term "about" includes not only the exact number recited after the term, but also to a range substantially or close to that number. Considering the context in which the number is presented, it can be determined whether the number is close to or near the specific number mentioned. As an example, the term “about” may refer to a range of -10% to +10% of a numeric value. As another example, the term “about” may refer to a range from -5% to +5% of a given numerical value. However, it is not limited thereto.

In the method for purifying the hyaluronidase, step (b) is a step of performing cation exchange chromatography (AEX).

Ion exchange chromatography refers to an exchange resin using the reversible interaction of the net charge between the medium and the protein surface, that is, the difference in ionic bond strength. Cation exchange chromatography used in the present invention means chromatography using a column filled with a cation exchange resin, and in the above step, cation exchange chromatography can be performed to further remove impurities and isoforms having a desired isoelectric point can be selectively separated.

The cation exchange resin refers to a synthetic resin that is added to another aqueous solution to exchange a specific cation in the aqueous solution with its own cation, and the cation exchange column can adsorb a protein having a cation above its isoelectric point.

In one embodiment, the functional groups of the cation exchange chromatography resin are methylsulfonate (S), sulfopropyl (SP), carboxymethyl (CM), poly aspartic acid, sulfoethyl (SE), sulfopropyl (SP), phosphate (P) or sulfonate (S) may be one selected from the group consisting of.

For example, the column that can be used for the cation exchange chromatography of the present invention is capto sulfopropyl (Capto SP), mono methyl sulfonate (Mono S), sulfopropyl sepharose (SP sepharose), sulfopropyl high performance (SP HP; SP High performance) or carboxymethyl sepharose (CM sepharose) may be used, and specifically, sulfopropyl sepharose (SP sepharose) may be used, but is not limited as long as it can isolate the desired hyaluronidase.

For example, as the column buffer of the cation exchange chromatography, a phosphate buffer, a citric acid buffer, or an acetate buffer may be used. For example, the column buffer may be a phosphate buffer, for example, sodium phosphate (Na ₂ HPO ₄ ). For example, the concentration of the column buffer may be adjusted to about 1 mM to 100 mM, for example, 20 mM to 80 mM, or 30 mM to 70 mM. For example, as the buffer, a buffer of pH 5 to 7 may be used. In one example, the flow rate may be about 0.1 ml/min to 15.0 ml/min, about 1 ml/min to 10.0 ml/min, or about 3.0 ml/min to 8.0 ml/min. However, it is not limited thereto.

For the purpose of the present invention, cation exchange chromatography may be to perform any one or more steps of a sample injection step, an equilibration step, a washing step, and an elution step.

The chromatography of the present invention is characterized in that it does not contain an additional stabilizer other than the buffer. The stabilizer is the same as described above.

In step (a) and/or (b), conditions such as flow rate, temperature, and time for flowing the solution may be appropriately adjusted.

For example, as the column buffer, a glycine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer may be used. Specifically, in step (a), a glycine buffer may be used, and in step (b), a phosphate buffer may be used, but is not limited thereto.

Steps (a) and/or (b) may use a concentration gradient. For example, the hyaluronidase can be eluted with a stepwise salt gradient or a continuous salt gradient.

Specifically, in the elution step of steps (a) and/or (b), an increasing concentration gradient (ascending gradient) of the buffer may be used. For example, a buffer having a concentration gradient in the range of about 0.0M to about 5.0M, about 0.0 to about 4.0M, about 0.0M to about 3.5M, and about 0.0M to about 3.0M may be used. The buffer may be, for example, sodium sulfate (Na ₂ SO ₄ ), sodium chloride (NaCl), potassium chloride (KCl), ammonium acetate (NH ₄ OAc), etc., specifically sodium chloride (NaCl), but is not limited thereto. It can be used by appropriately selecting from a range known in the art.

In the method for purifying the hyaluronidase, step (c) is a step of performing size exclusion chromatography (SEC).

The size exclusion chromatography used in the present invention means to separate a mixture based on the rate (permeability) at which solutes of various sizes pass through a porous matrix. In size exclusion chromatography, when an analyte sample is passed through a column filled with a porous stationary phase such as a gel, matrix, or beads, large molecules that cannot pass through the pores of the column cannot enter the pores. This may be due to the principle that small molecules exit the column by moving relatively slowly as they exit through the hole of the column, while rapidly exiting the column through the empty space.

In one embodiment, the column that can be used for size exclusion chromatography of the present invention is Superdex, Sephacryl, Superose, Sephadex, Sepharose, Polyacryl Amide (polyacrylamide) or silica-based (silica-based) column may be used, and specifically may be sepacryl, but is not limited as long as it can separate the desired hyaluronidase.

For example, a phosphate buffer, a citric acid buffer, or an acetate buffer may be used as the column buffer for the size exclusion chromatography. For example, the column buffer may be a citrate buffer, for example, sodium citrate. For example, the concentration of the column buffer may be adjusted to about 1 mM to 100 mM, for example, 5 mM to 50 mM, or 10 mM to 30 mM. For example, as the buffer, a buffer of pH 4 to 6 may be used. In one example, the flow rate may be from about 0.001 ml/min to 5.0 ml/min, from about 0.1 ml/min to 3.0 ml/min, or from about 0.1 ml/min to 1.0 ml/min. However, it is not limited thereto.

In any one of the above embodiments, the hyaluronidase may be separated through the size exclusion chromatography step of the present invention, but is not limited thereto.

For the purpose of the present invention, size exclusion chromatography may be to perform any one or more steps of a sample injection step, an equilibration step, a washing step, and an elution step.

The chromatography of the present invention is characterized in that it does not contain an additional stabilizer other than the buffer. The stabilizer is the same as described above.

For the purpose of the present invention, the hyaluronidase obtained from the chromatography of each step may have high stability and purity even if it does not contain the additional stabilizer as described above.

As an example, even if an additional stabilizer is not included in the process of the purification method of the present invention, affinity chromatography (AC) -> cation exchange chromatography (CEX) -> size exclusion chromatography (SEC), It was confirmed that the purity of the hyaluronidase was the highest without the inclusion of impurities.

This suggests that the purification method of the present invention can obtain high-purity hyaluronidase without adding an additional stabilizer, and thus is an excellent method in terms of economics including time and cost.

After the hyaluronidase purification method of steps (a) to (c) of the present invention, that is, a three-step column process, the hyaluronidase of high purity and high yield from which impurities are efficiently removed can be purified.

In the present invention, any one of a concentration and dialysis process and a filtration process may be additionally performed after steps (a) to (c), but the present invention is not limited thereto.

The filtration process may be carried out in a process such as conventionally known microfiltration, ultrafiltration, microfiltration, depth filtration, but is not limited thereto.

The hyaluronidase isolated according to the purification method of the present invention may have high purity. Specifically, it may have a purity of about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 97.5% or more, or about 98% or more, but is not limited thereto.

In addition, the purity may simply indicate the purity of the material separated from the elution solution, but the % of the final purity may vary depending on the % of the purity of the loaded sample.

In addition, the purity of the hyaluronidase may be analyzed through HPLC or SDS-PAGE analysis after purification from the elution solution, but is not limited thereto.

For example, when the purification method of the present invention, affinity chromatography (AC) -> cation exchange chromatography (CEX) -> size exclusion chromatography (SEC), was performed in the order, it was confirmed that the final yield was the best. . This suggests that even if three identical chromatography (affinity chromatography, cation exchange chromatography, and size exclusion chromatography) are used, there is a difference in purification efficiency depending on how the process sequence is performed.

By using the purification method of the present invention, high-purity hyaluronidase can be obtained economically and efficiently.

1 shows the results of SDS-PAGE of purified hyaluronidase.
2 shows the Western-Blot results of purified hyaluronidase.
3 shows the results of comparison and analysis of purity by changing the order of the purification process of the present invention.
4 shows the results of comparative analysis of the purity and the process including the stabilizer in the buffer during the purification process of the present invention.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1. Hyaluronidase (Hyaluronidase, HAdase) primary purification

1-1. Blue Sepharose Chromatography

A loading sample containing hyaluronidase was prepared for primary purification using Blue Sepharose resin. Specifically, it was loaded onto a column filled with blue sepharose resin dissolved in 20 mM glycine (Glycine), pH 10.0 buffer. Equilibration/washing was performed by flowing the equilibration/elution buffer (20mM glycine, pH 10.0) before the loading at a flow rate of 7ml/min and a conductivity of 1mS/cm.

After loading the solution containing hyaluronidase on the Blue Sepharose column, it was washed with about 10 column volumes of 20 mM glycine, pH 10.0 buffer, and then increased using pH 10.0 buffer containing 20 mM glycine and 1 M sodium chloride (NaCl). The hyaluronidase was eluted from the column via a salt step change.

1-2. Eluted Sample Dilution

The eluate collected by the method of Example 1-1 was diluted with 50 mM sodium phosphate buffer, pH 6.0, and then adjusted to pH 6.0 and conductivity 5 mS/cm or less.

Example 2. Secondary purification of hyaluronidase

2-1. Cation exchange resin chromatography

The diluted eluent of Example 1-2 was injected into FPLC (Fast Protein Liquid Chromatography) connected to an SP Sepharose column, AKTA Prime Plus.

Specifically, before injection of the supernatant, sodium phosphate (Na ₂ HPO ₄ ) buffer (50 mM, pH 6.0) was flowed into a column filled with SP Sepharose resin to equilibrate, and the sodium phosphate buffer solution containing the hyaluronidase (50 mM) , pH 6.0) was flowed through the column. After equilibration, the injection step was performed and the flow through (FT) liquid was collected in a sterile vessel while the hyaluronidase was bound to the cation exchange column material. This was followed by a wash step, in which at least about 10 column volumes of wash buffer (50 mM sodium phosphate, pH 6.0) were passed through the cation exchange column. The next step was the elution step, which was followed by washing with at least about 10 column volumes of 50 mM sodium phosphate, pH 6.0 buffer and then from the column through incremental salt step changes using pH 6.0 buffer containing 50 mM sodium phosphate, 1 M sodium chloride. Hyaluronidase was eluted.

Cation exchange resin chromatography was performed under the conditions of a conductivity of 4 mS/cm and a flow rate of 6 ml/min.

2-2. Eluted Sample Concentration

The eluent collected by the method of Example 2-1 was concentrated using Amicon-Ultra-15, Centrifugal Filter (10K). Centrifugation was carried out at 6000 rpm for 30 min each, and concentrated until the final eluent volume reached 3 ml, and then filtered through PVDF 0.22 μm (Millipore Millex-GV) without contact with the outside.

Example 3. Hyaluronidase tertiary purification

3-1. size exclusion chromatography

The concentrated eluent of Example 2-2 was loaded onto a Sephacryl S-200 HR column-connected FPLC (high-speed protein liquid chromatography). The column was equilibrated by flowing an equilibration/elution buffer (20 mM sodium citrate, Na ₂ HC ₆ H ₅ O _{7 ), pH 5.0 before the loading.} After equilibration, 20 mM sodium citrate buffer at pH 5.0 was flowed at a flow rate of 0.5 ml/min, and fractions of 50 mAu or more were sequentially obtained at UV 280 nm wavelength among the eluted peaks. After the fractions were obtained, the column was washed by injecting 20 mM sodium citrate, 1 M sodium chloride, pH 5.0 as a washing buffer for regeneration of the column.

Example 4. Hyaluronidase Assay

4-1. Analysis of high-purity hyaluronidase according to the purification method of the present invention

The activity of purified hyaluronidase according to Examples 1 to 3 was evaluated. Activity was measured according to the assay of the United States Pharmacopoeia "USP assay for Hyaluronidase". As a result of the analysis using the above evaluation method, it was confirmed that the activity of the loading sample before purification was 2,051 unit/mg, and the specific activity of the high-purity hyaluronidase after purification was 273,840 unit/mg.

In addition, as a result of SDS-PAGE of high-purity hyaluronidase, only a band identical to the molecular weight (2 band) of previously known hyaluronidase was detected (Robin AP HARRISON, Biochem. J. 1988, 252, 875-882), It was confirmed that no impurities were detected with a purity of 98% or more (FIG. 1).

For Western-Blot analysis, abcam's Anti-SPAM1 antibody [1A4], (ab50694), an antibody that binds only to hyaluronidase, was used, and Western Blot results also confirmed that both bands were hyaluronidase ( 2).

As a result, when performing three steps in the order of affinity chromatography including a blue sepharose column, cation chromatography including a SP sepharose column, and size exclusion chromatography, hyaluronidase from which impurities are almost removed This suggests that it can be purified with high purity.

4-2. Analysis of hyaluronidase according to comparative purification method

In order to confirm whether the high-purity hyaluronidase was purified when the process was performed in the order of the purification method of the present invention, the results according to the change in the order of the primary and secondary purification processes were compared. Specifically, three steps were performed in the order of cation chromatography including an SP sepharose column, affinity chromatography including a blue sepharose column, and size exclusion chromatography.

As a result, as shown in FIG. 3, when the order of the purification process is changed, it can be seen that the purity is lower than the result of the purification method of the present invention.

Through this, it can be seen that the process sequence of the purification method is an important factor in order to obtain high-purity hyaluronidase even if the same chromatography is used.

4-3. Analysis of hyaluronidase with addition of stabilizer

In the case of additionally adding a stabilizer in the purification method of the present invention, in order to check whether high-purity hyaluronidase is purified, a stabilizer containing 1 mM EDTA and 0.1% Tween 80 is added when the SP Sepharose column is performed, A stabilizing agent comprising 1 mM magnesium chloride (MgCl ₂ ) and 0.01% Tween 80 was additionally added when size exclusion chromatography was performed.

As a result, as shown in FIG. 4 , it can be confirmed that impurities appear when a stabilizer is additionally added, and it can be seen that the purity is lower than that of the purification method of the present invention in which a stabilizer is not added. .

Through this, the purification method of the present invention can obtain high-purity hyaluronidase without adding an additional stabilizer.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (12)

As a method for purifying hyaluronidase, the method is to purify a hyaluronidase-containing raw material in the following order, as a purification method, the following steps (a) to (c) are performed once and, in addition to not performing the same or different chromatography, the purification method:
(a) performing affinity chromatography (AC);
(b) performing cation exchange chromatography (CEX) using sulfopropyl sepharose (SP sepharose); and
(c) performing size exclusion chromatography (SEC).
delete According to claim 1,
The other chromatography is one or more selected from the group consisting of dye affinity chromatography, reverse phase chromatography, and anion exchange chromatography, the purification method.
According to claim 1,
Affinity chromatography of step (a) is Blue Sepharose, Blue Sepharose FF (Blue Sepharose Fast Flow), Heparin Sepharose HP (Heparin Sepharose High Performance) or Heparin Sepharose FF (Heparin Sepharose Fast Flow) ) is to use a column, the purification method.
delete According to claim 1,
The size exclusion chromatography of step (c) is based on Superdex, Sephacryl, Superose, Sephadex, Sepharose, Polyacrylamide or silica (silica-based) of the use of a column, the purification method.
According to claim 1,
The chromatography of each step is to further perform any one or more steps of a sample injection step, an equilibration step, a washing step, and an elution step.
According to claim 1,
The (a) step is to be carried out using a buffer containing 1 to 50 mM of glycine and having a pH of 8.0 to 12.0, the purification method.
According to claim 1,
The step (b) is 1-75 mM sodium phosphate (Na ₂ HPO ₄ ) It is carried out using a buffer having a pH of 5.0 to 7.0, the purification method.
According to claim 1,
The (c) step is 1 to 50 mM sodium citrate (Na ₂ HC ₆ H ₅ O ₇ ) The purification method is performed using a buffer having a pH of 4.0 to 6.0.
11. The method according to any one of claims 8 to 10,
The method does not include an additional stabilizer other than the buffer of each step, the purification method.
According to claim 1,
The hyaluronidase purified by the above method will have a purity of 98% or more.

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