Hyaluronidase and process for its preparation

Abstract

The application belongs to the technical field of biology and relates to a hyaluronidase and a preparation process thereof. The preparation process of the hyaluronidase comprises the following steps: (1) extracting a fresh bovine testis after homogenization to obtain an intermediate I; (2) purifying the intermediate I through a hydrophobic chromatography column to obtain an intermediate II; (3) purifying the intermediate II through a cation exchange chromatography column to obtain an intermediate III; and (4) freeze-drying the intermediate III to obtain a hyaluronidase product. The method has the advantages of simple operation, low production cost, high product yield and higher purity of the hyaluronidase.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology and relates to a hyaluronidase and a preparation process thereof. Background Art

[0002] Hyaluronidase (HAase), also known as hyaluronidase, is an endoglycosidase extracted from the testicles of mammals such as cattle and sheep or produced by microbial fermentation. It degrades hyaluronic acid by acting on β-1,3 or β-1,4 glycosidic bonds. It also has a catalytic effect on chondroitin and chondroitin sulfate to a certain extent.

[0003] Hyaluronidase can temporarily reduce the viscosity of the intercellular matrix when applied to the human body, accelerating the diffusion of subcutaneous infusions, locally accumulated exudates, or blood, thereby facilitating absorption. It is an important drug diffusion agent. Clinically, it is used as a drug penetrant to promote drug absorption and the dissipation of local edema or hematoma after surgery and trauma.

[0004] The 2020 edition of the Chinese Pharmacopoeia stipulates that hyaluronidase is an enzyme extracted from mammalian testicles that hydrolyzes hyaluronic acid mucopolysaccharides. The activity of hyaluronidase per 1 mg of protein must not be less than 300 units. The activity of hyaluronidase per 1 mg of protein must not be less than 1800 units. Currently, the preparation process for hyaluronidase mainly consists of two stages: extraction and purification. The extraction solvents used in the extraction stage are mostly acetic acid, sodium acetate buffer, and sodium phosphate buffer. After extraction, ammonium sulfate salting-out or ethanol precipitation is performed to remove some impurities. This process requires the use of large amounts of salt or organic solvents. Salting-out and ethanol precipitation also have low resolution, resulting in significant product loss and difficulty in ensuring yield. The most commonly used techniques in the purification stage are ion exchange chromatography and gel filtration chromatography. Ion exchange chromatography offers the advantages of high throughput and resolution. Gel filtration chromatography utilizes the molecular sieving effect of the network-like structure of gels to separate substances based on molecular weight, making it an ideal technique for fine purification. However, this method has limited throughput, requires multiple chromatographic operations, and results in long processing times and low production efficiency. Therefore, it is necessary to research and develop new preparation processes to improve production efficiency and product yield and reduce production costs while ensuring product purity. Summary of the Invention

[0005] In response to the current purification problem, the purpose of this application is to provide a method for preparing hyaluronidase. Compared with existing processes, this preparation method is simple to operate, has low production cost, and obtains hyaluronidase with higher purity.

[0006] The present application provides a preparation process of hyaluronidase, comprising the following steps:

[0007] (1) Fresh bovine testicles are homogenized and extracted to obtain intermediate I;

[0008] (2) the intermediate I is purified by hydrophobic chromatography column to obtain intermediate II;

[0009] (3) the intermediate II is purified by cation exchange chromatography column to obtain intermediate III;

[0010] (4) the intermediate III is freeze-dried to obtain hyaluronidase product.

[0011] In some embodiments of the present application, the step (1) comprises the following steps: fresh bovine testis is washed and stripped of outer membrane, chopped and homogenized by homogenizer, the homogenate is transferred to buffer 1, stirred and extracted for 2 hours, the supernatant is collected by centrifugation; the centrifugal precipitate is added with buffer 2, stirred and extracted for 2 hours, the supernatant is collected by centrifugation; the two supernatants are combined, which is intermediate I.

[0012] In some embodiments of the present application, the specific conditions of homogenization are as follows: rotation speed of 1000-1500 rpm, time of 3-5 min.

[0013] In some embodiments of the present application, in the step (1), the buffer 1 and the buffer 2 are both 50mM sodium acetate solution containing 50mM sodium chloride, and the pH is 5.0-5.5.

[0014] In some embodiments of the present application, the solid-liquid ratio of the fresh bovine testis and the buffer 1 is 1kg: (1.8-2.2) L.

[0015] In some embodiments of the present application, the mass ratio of the centrifugal precipitate and the buffer 2 is 1kg: (0.8-1.2) L.

[0016] In some embodiments of the present application, the rotation speed of stirring extraction is 80-100 rpm.

[0017] In some embodiments of the present application, the step (2) comprises the following steps: solid ammonium sulfate is added to the intermediate I, stirred and dissolved, the pH and conductivity are adjusted to be consistent with the equilibration buffer, and the sample is loaded into the equilibrated hydrophobic chromatography column; after the loading is completed, the equilibration buffer is used to wash the unabsorbed protein, and the absorbance value of the effluent at 280 nm is detected until the absorbance value no longer decreases; then the eluent is used for elution, and the absorbance value of the eluent at 280 nm is detected, and the elution peak is collected, which is intermediate II.

[0018] In some embodiments of the present application, in the step (2), the equilibration buffer is 0.05M sodium acetate solution containing 1.2M (NH4)2SO4, and the pH is 6.0.

[0019] In some embodiments of the present application, sodium acetate-sodium chloride solution is selected to collaboratively extract hyaluronidase, which can maintain enzyme activity, promote tissue dissolution, and suppress impurities, and contribute to the subsequent use of hydrophobic chromatography and cation exchange chromatography purification to obtain hyaluronidase with higher titer, specific activity and purity. This may be because on the one hand, the sodium acetate buffer system is close to the optimal pH of hyaluronidase, inhibits protease activity, and reduces enzyme degradation during the extraction process; on the other hand, 50mM sodium chloride destroys the connection between testicular tissue cells through the salt dissolution effect, improves the efficiency of homogenization, and avoids enzyme precipitation caused by high salt. In addition, the low salt concentration environment inhibits the dissolution of impurities such as nucleic acids and polysaccharides, simplifying the subsequent purification steps.

[0020] In some embodiments of the present application, in step (2), the filler of the hydrophobic chromatography column is a copolymer of vinyl alcohol and methacrylate with butyl, octyl, phenyl or neopentyl as a ligand, agarose, cellulose, polystyrene, polymethyl acrylate or chitosan.

[0021] In some embodiments of the present application, the hydrophobic chromatography column is Phenyl Phenyl-650M.

[0022] In some embodiments of the present application, hydrophobic chromatography is used instead of ammonium sulfate salting-out as a preliminary purification method, which can greatly improve the resolution and reduce product loss. This is because the hydrophobic chromatography fillers with ligands such as phenyl achieve separation through the difference in surface hydrophobicity between the target protein and the impurity protein. Hyaluronidase has a weak surface hydrophobicity and preferentially binds to the filler under high salt conditions, while the impurity protein is eluted due to its strong hydrophobicity. Traditional salting-out requires centrifugation or filtration to remove the precipitate, which can easily lead to co-precipitation or mechanical loss of the target protein. Hydrophobic chromatography accurately separates through gradient elution, greatly improving the recovery rate.

[0023] In some embodiments of the present application, in step (2), the eluent is a 0.05 M sodium acetate solution containing 0.35-0.4 M (NH4)2SO4, with a pH of 6.0.

[0024] In some embodiments of the present application, step (3) includes the following steps: intermediate II is ultrafiltered with an ultrafiltration membrane with a molecular weight cutoff of 10kD, and then ultrafiltration salt is exchanged to make the pH and conductivity of the ultrafiltrate consistent with the equilibrium solution, and the sample is loaded onto a cation exchange chromatography column after dissolution, and the chromatography column is pre-equilibrated with the equilibrium solution; after the loading is completed, the unadsorbed protein is washed with the equilibrium solution, and the absorbance of the washing solution at 280nm is detected until the absorbance value no longer decreases; then the intermediate is eluted with an eluent, and the absorbance of the eluent at 280nm is detected until the absorbance value no longer decreases, and the elution peak is collected, which is intermediate III.

[0025] In some embodiments of the present application, in step (3), the solution and the equilibrium solution used for ultrafiltration salt exchange are both 0.1M NaAc, with a pH of 5.5.

[0026] In some embodiments of the present application, in step (3), the ligand of the cation exchange chromatography column filler is carboxymethyl, sulfonic acid, phosphate, phosphite, or phenol.

[0027] In some embodiments of the present application, the cation exchange chromatography column is an SP-Sepharose chromatography column.

[0028] In some embodiments of the present application, in step (3), the eluent is a 0.1 M NaAc and 0.1 M NaCl solution with a pH of 4.5-5.0.

[0029] In some embodiments of the present application, by regulating the pH of the solution, optimizing the separation efficiency of cation exchange chromatography, utilizing the amphoteric ionization properties of proteins, and changing the pH of the solution to make the proteins carry different charges, the titer and specific activity of hyaluronidase are improved. This may be because proteins are amphoteric molecules, and their charge changes with pH. When the pH of the solution is lower than the isoelectric point of hyaluronidase, the enzyme is positively charged and can bind to cation exchange fillers with ligands such as carboxymethyl, and impurity proteins are eluted due to pI differences or insufficient charge. A specific eluent linear gradient elution is used, and the salt concentration gradient is used to destroy the electrostatic interaction between the enzyme and the filler, thereby achieving high-purity collection and improving the specific activity of hyaluronidase.

[0030] In some embodiments of the present application, the preparation process further comprises ultrafiltration of the intermediate III obtained in step (3) to exchange salt, and the solution used for ultrafiltration to exchange salt is 0.01M sodium acetate buffer solution with a pH of 6.0-7.0.

[0031] In some embodiments of the present application, ultrafiltration salt exchange is introduced after cation exchange chromatography to improve enzyme activity and the stability of the freeze-dried product. This may be because the ultrafiltration membrane intercepts large molecular impurities and replaces ammonium sulfate, NaCl, etc. in the chromatography buffer with a low-salt buffer, reducing the inhibition of enzyme activity by ionic strength, removing impurity ions, and ensuring enzyme activity. Adjusting the pH to 6.0-7.0 (close to neutral) before freeze-drying avoids enzyme denaturation caused by acidic or alkaline conditions and improves the stability of the freeze-dried product. In addition, the ultrafiltration concentration process can further remove small molecular impurities and improve the final purity of hyaluronidase.

[0032] In some embodiments of the present application, step (4) includes the following steps: concentrating the intermediate III by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 10 kD, ultrafiltration to exchange salt, and freeze-drying to obtain the finished hyaluronidase product.

[0033] Compared with the prior art, the present application at least obtains the following technical effects:

[0034] The present application uses bovine testis as the starting material, and hyaluronidase is prepared by sodium acetate-sodium chloride solution extraction, followed by hydrophobic chromatography and cation exchange chromatography purification. In the hydrophobic chromatography, the target protein is separated from impurities based on the difference in hydrophobicity, which can greatly improve the resolution and reduce product loss compared with ammonium sulfate salting-out. By utilizing the amphoteric ionization property of proteins, different charges are carried on the proteins by changing the pH of the solution, and the hyaluronidase product titer reaches more than 1600 IU / mg and the specific activity reaches more than 3300 IU / mg of protein after cation exchange chromatography. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the process flow chart of the present application;

[0036] Figure 2 is the HPLC purity chromatogram of the hyaluronidase prepared in Example 1;

[0037] Figure 3 is the HPLC purity chromatogram of the hyaluronidase prepared in Example 2;

[0038] Figure 4 is the HPLC purity chromatogram of the hyaluronidase prepared in Comparative Example 1;

[0039] Figure 5 is the HPLC purity chromatogram of the hyaluronidase prepared in Comparative Example 2;

[0040] Figure 6 is the HPLC purity chromatogram of the hyaluronidase prepared in Comparative Example 3;

[0041] Figure 7 is the HPLC purity chromatogram of the hyaluronidase prepared in Comparative Example 4;

[0042] Figure 8 is the HPLC purity chromatogram of the hyaluronidase prepared in Comparative Example 5. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present application more clear and obvious, the present application will be further described in detail below. However, it should be understood that the description here is only used to explain the present application, and is not used to limit the scope of the present application.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available. The characterization methods involved can be found in the relevant descriptions in the prior art and will not be described in detail herein.

[0045] Example 1

[0046] This embodiment provides a preparation process of hyaluronidase, and the specific steps are as follows: Figure 1 As shown:

[0047] (1) 20 kg of fresh bovine testicles were cleaned and the outer membranes were removed. The testicles were minced and homogenized using a homogenizer. The homogenate was transferred to 40 L of buffer 1 and stirred for 2 h. The supernatant was collected by centrifugation. The centrifuged precipitate was added to 20 L of buffer 2 and stirred for 2 h. The supernatant was collected by centrifugation. The two supernatants were combined to obtain intermediate I.

[0048] (2) Add solid ammonium sulfate to intermediate I, stir and dissolve, adjust the pH and conductivity to be consistent with the equilibrium solution, and load the sample onto the equilibrated hydrophobic chromatography column; after loading, wash the unadsorbed protein with the equilibrium solution and detect the absorbance of the effluent at 280 nm until the absorbance no longer decreases; then elute with the eluent and detect the absorbance of the eluent at 280 nm. Collect the elution peak, which is intermediate II;

[0049] (3) Intermediate II is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kD and then ultrafiltered to exchange salt until the pH and conductivity of the ultrafiltrate are consistent with those of the equilibrium solution. After dissolution, the sample is loaded onto a cation exchange chromatography column, which has been pre-equilibrated with the equilibrium solution. After loading, the unadsorbed protein is washed with the equilibrium solution, and the absorbance of the washing solution at 280 nm is detected until the absorbance no longer decreases. The protein is then eluted with the eluent, and the absorbance of the eluent at 280 nm is detected until the absorbance no longer decreases. The elution peak is collected, which is intermediate III.

[0050] (4) After ultrafiltration and concentration of intermediate III using an ultrafiltration membrane with a molecular weight cutoff of 10 kD, ultrafiltration was performed using a 0.01 M sodium acetate buffer solution with a pH of 7.0 to exchange salt, and the hyaluronidase product was obtained after freeze-drying.

[0051] The specific conditions of the homogenization are: rotation speed 1000 rpm, time 3 min.

[0052] The rotation speed of the stirring extraction is 80 rpm.

[0053] In step (1), buffer 1 and buffer 2 are both 50 mM sodium acetate solutions containing 50 mM sodium chloride, with a pH of 5.5.

[0054] In step (2), the equilibrium solution is a 0.05M sodium acetate solution containing 1.2M (NH4)2SO4, with a pH of 6.0.

[0055] The hydrophobic chromatography column is Phenyl-650M, with a column volume of 10 L and a diameter of 20 cm.

[0056] In step (2), the eluent is a 0.05 M sodium acetate solution containing 0.35 M (NH 4 ) 2 SO 4 , with a pH of 6.0.

[0057] In step (3), the solution and the equilibrium solution used for ultrafiltration salt exchange are both 0.1M NaAc, with a pH of 5.5.

[0058] In step (3), the cation exchange chromatography column is an SP-Sepharose chromatography column with a column volume of 1 L and a diameter of 7.5 cm.

[0059] In step (3), the eluent is a 0.1 M NaAc solution containing 0.1 M NaCl, with a pH of 4.5.

[0060] The relevant parameters of hyaluronidase finished product are shown in Table 1. HPLC purity spectrum is shown in Figure 2 As shown; chromatographic results, see Table 2.

[0061] Table 1 Parameters of hyaluronidase finished product prepared in Example 1

[0062]

[0063] Table 2 Chromatographic results

[0064]

[0065] From Table 1, Figure 2 As shown in Table 2, the titer and specific activity of the hyaluronidase prepared by the present process exceed the requirements of the Chinese Pharmacopoeia (the activity of hyaluronidase must not be less than 300 units per 1 mg of hyaluronidase, and the activity of hyaluronidase must not be less than 1800 units per 1 mg of protein). The tyrosine content is well below the Chinese Pharmacopoeia limit (the tyrosine content must not exceed 0.1 μg per unit of hyaluronidase), and the product yield reaches 73.6%. HPLC analysis shows a purity of 99.84%, with a peak resolution of 4.6, indicating that the product can be completely separated from impurities and that the analytical method is feasible.

[0066] Example 2

[0067] This embodiment provides a preparation process of hyaluronidase, and the specific steps are as follows:

[0068] (1) 40 kg of fresh bovine testicles were cleaned and the outer membranes were removed. The testicles were minced and homogenized using a homogenizer. The homogenate was transferred to 80 L of buffer 1 and stirred for 2 h. The supernatant was collected by centrifugation. The centrifuged precipitate was added to 40 L of buffer 2 and stirred for 2 h. The supernatant was collected by centrifugation. The two supernatants were combined to obtain intermediate I.

[0069] (2) Add solid ammonium sulfate to intermediate I, stir and dissolve, adjust the pH and conductivity to be consistent with the equilibrium solution, and load the sample onto the equilibrated hydrophobic chromatography column; after loading, wash the unadsorbed protein with the equilibrium solution and detect the absorbance of the effluent at 280 nm until the absorbance no longer decreases; then elute with the eluent and detect the absorbance of the eluent at 280 nm. Collect the elution peak, which is intermediate II;

[0070] (3) Intermediate II is ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kD and then ultrafiltered to exchange salt until the pH and conductivity of the ultrafiltrate are consistent with those of the equilibrium solution. After dissolution, the sample is loaded onto a cation exchange chromatography column, which has been pre-equilibrated with the equilibrium solution. After loading, the unadsorbed protein is washed with the equilibrium solution, and the absorbance of the washing solution at 280 nm is detected until the absorbance no longer decreases. The protein is then eluted with the eluent, and the absorbance of the eluent at 280 nm is detected until the absorbance no longer decreases. The elution peak is collected, which is intermediate III.

[0071] (4) After ultrafiltration and concentration of intermediate III using an ultrafiltration membrane with a molecular weight cutoff of 10 kD, ultrafiltration was performed using a 0.01 M sodium acetate buffer solution with a pH of 6.0 to exchange salt, and the hyaluronidase product was obtained after freeze-drying.

[0072] The specific conditions of the homogenization are: rotation speed 1500 rpm, time 5 min.

[0073] The rotation speed of the stirring extraction is 100 rpm.

[0074] In step (1), buffer 1 and buffer 2 are both 50 mM sodium acetate solutions containing 50 mM sodium chloride, with a pH of 5.0.

[0075] In step (2), the equilibrium solution is 0.05M sodium acetate and 1.2M (NH4)2SO4 solution, with a pH of 6.0.

[0076] The hydrophobic chromatography column is Phenyl-650M, with a column volume of 20 L and a diameter of 30 cm.

[0077] In step (2), the eluent is 0.05M sodium acetate and 0.4M (NH4)2SO4 solution, with a pH of 6.0.

[0078] In step (3), the solution and the equilibrium solution used for ultrafiltration salt exchange are both 0.1M NaAc, with a pH of 5.5.

[0079] In step (3), the cation exchange chromatography column is an SP-Sepharose chromatography column with a column volume of 2 L and a diameter of 10 cm.

[0080] In step (3), the eluent is a 0.1 M NaAc solution containing 0.1 M NaCl, with a pH of 5.0.

[0081] The relevant parameters of the finished hyaluronidase are shown in Table 3. HPLC purity profile is shown in Figure 3 As shown; chromatographic results, see Table 4.

[0082] Table 3 Parameters of hyaluronidase finished product prepared in Example 2

[0083]

[0084] Table 4 Chromatographic results

[0085]

[0086] From Table 3, Figure 3 As can be seen from Table 4, the titer and specific activity of hyaluronidase prepared by the process of the present invention are higher than the requirements of the Chinese Pharmacopoeia (the activity of hyaluronidase per 1 mg shall not be less than 300 units, and the activity of hyaluronidase per 1 mg of protein shall not be less than 1800 units), the tyrosine content is far below the limit of the Chinese Pharmacopoeia (the tyrosine content per unit of hyaluronidase shall not exceed 0.1 μg), and the product yield reaches 72.7%.

[0087] HPLC analysis showed a purity of 99.75% and a product peak separation of 4.6, indicating that the product and impurities can be completely separated and the analytical method is feasible. The results of the hyaluronidase prepared in Example 2 were consistent with those in Example 1, indicating that the process is stable and repeatable.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 1 is:

[0090] In step (3), the eluent is a 0.1 M NaAc solution containing 0.1 M NaCl, with a pH of 6.0.

[0091] The relevant parameters of the hyaluronidase finished product are shown in Table 5. HPLC purity profile is shown in Figure 4 As shown; chromatographic results, see Table 6.

[0092] Table 5 Parameters of hyaluronidase finished product prepared in Comparative Example 1

[0093]

[0094] Table 6 Chromatographic results

[0095]

[0096] From Table 5, Figure 4 As shown in Table 6, after the pH of the eluent in step 3 of the present invention is adjusted to 6.0, the yield of the obtained product is basically unaffected, but the product titer and specific activity are reduced. Figure 4 It can be seen that the impurities in the product increased significantly, and the product purity dropped from more than 99% in Example 1 and Example 2 to 92.57%, indicating that the solution pH affects the product purification effect during cation exchange chromatography.

[0097] Comparative Example 2

[0098] The difference between this comparative example and Example 1 is that the hydrophobic chromatography column in step (2) is replaced by a dextran gel column (Sephadex G-75, column diameter 10 cm, column volume 4.7 L).

[0099] The relevant parameters of the hyaluronidase finished product are shown in Table 7. HPLC purity profile is shown in Figure 5 As shown; chromatographic results, see Table 8.

[0100] Table 7 Parameters of hyaluronidase finished product prepared in Comparative Example 2

[0101] Table 8 Chromatographic results

[0102]

[0103] From Table 7, Figure 5 As shown in Table 8, after the hydrophobic chromatography in the process of the present invention was replaced by Sephadex G-75 gel filtration chromatography, the yield, titer and specific activity of the obtained product were greatly reduced. Figure 5 It can be seen that the impurities in the product increased significantly, and the product purity dropped from more than 99% in Examples 1 and 2 to 69.54%, indicating that the choice of chromatography type will affect the product purification effect.

[0104] Comparative Example 3

[0105] The difference between this comparative example and Example 1 is that both buffer solution 1 and buffer solution 2 are 0.1 M acetic acid-sodium acetate solutions containing 0.3 M sodium chloride.

[0106] The relevant parameters of the hyaluronidase finished product are shown in Table 9. HPLC purity profile is shown in Figure 6 As shown; the chromatographic results are shown in Table 10.

[0107] Table 9 Parameters of hyaluronidase finished product prepared in Comparative Example 3

[0108]

[0109] Table 10 Chromatographic results

[0110]

[0111] From Table 9, Figure 6 As shown in Table 10, after the extraction buffer in the process of the present invention was adjusted to a 0.1 M acetic acid-sodium acetate solution containing 0.3 M sodium chloride, the yield, titer, and specific activity of the obtained product were all reduced, and the product purity decreased from more than 99% in Examples 1 and 2 to 95.73%, indicating that the extraction solution affects the product extraction and subsequent purification effects.

[0112] Comparative Example 4

[0113] The difference between this comparative example and Example 1 is that the cation exchange chromatography filler is CM-Sephadex A50.

[0114] The relevant parameters of hyaluronidase finished product are shown in Table 11. HPLC purity spectrum is shown in Figure 7 As shown; chromatographic results, see Table 12.

[0115] Table 11 Hyaluronidase finished product parameters prepared in Comparative Example 4

[0116]

[0117] Table 12 Chromatographic results

[0118]

[0119] From Table 11, Figure 7 As shown in Table 12, after the cationic chromatography filler in the process of the present invention was adjusted to CM-Sephadex A50, the yield, potency, and specific activity of the obtained product were reduced, and the product purity decreased from more than 99% in Examples 1 and 2 to 93.54%, indicating that the type of cationic chromatography filler can affect the product purification effect.

[0120] Comparative Example 5

[0121] The difference between this comparative example and Example 1 is that the hydrophobic chromatography filler is Butyl Sepharose 4FF.

[0122] The relevant parameters of the finished hyaluronidase are shown in Table 13. The HPLC purity profile is shown in Figure 8 As shown; chromatographic results, see Table 14.

[0123] Table 13 Hyaluronidase finished product parameters prepared in Comparative Example 4

[0124]

[0125] Table 14 Chromatographic results

[0126]

[0127] From Table 13, Figure 8 As shown in Table 14, after the hydrophobic chromatography filler in the process of the present invention was adjusted to Butyl Sepharose 4FF, the yield, titer, and specific activity of the obtained product were reduced, and the product purity decreased from more than 99% in Examples 1 and 2 to 91.34%, indicating that the type of hydrophobic chromatography filler can affect the product purification effect.

[0128] The applicant declares that this application uses the above-mentioned examples to illustrate the preparation process of a hyaluronidase of this application, but this application is not limited to the above-mentioned examples, that is, it does not mean that this application must rely on the above-mentioned examples to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent replacement of various raw materials of the product of this application, addition of auxiliary ingredients, selection of specific methods, etc., all fall within the scope of protection and disclosure of this application.

[0129] The preferred embodiments of the present application are described in detail above. However, the present application is not limited to the specific details of the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the scope of protection of the present application.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner unless there is any contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

Claims

1. A preparation process of hyaluronidase, characterized in that: The following steps are involved: (1) Wash the fresh bovine testicles, remove the outer membrane, mince them, and homogenize them with a homogenizer. Transfer the homogenate to buffer 1, stir and extract for 2 hours, and collect the supernatant by centrifugation. The centrifuged precipitate was added with buffer 2, stirred and extracted for 2 h, and the supernatant was collected by centrifugation; the two supernatants were combined to obtain intermediate I; buffer 1 and buffer 2 were both 50 mM sodium acetate solutions containing 50 mM sodium chloride, with a pH of 5.0-5.5; (2) Add solid ammonium sulfate to intermediate I, stir to dissolve, adjust the pH and conductivity to be consistent with the equilibrium solution, and load the sample onto the equilibrated hydrophobic chromatography column; After loading, the unadsorbed protein is washed with the equilibration solution, and the absorbance of the effluent at 280 nm is detected until the absorbance no longer decreases; the protein is then eluted with the eluent, and the absorbance of the eluent at 280 nm is detected, and the elution peak is collected, which is intermediate II; the filler of the hydrophobic chromatography column is a copolymer of vinyl alcohol and methacrylate with butyl, octyl, phenyl or neopentyl groups as ligands, agarose, cellulose, polystyrene, polymethyl acrylate or chitosan; (3) Intermediate II was ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 10 kD and then the ultrafiltration salt was exchanged to make the pH and conductivity of the ultrafiltrate consistent with the equilibrium solution. After dissolution, the sample was loaded onto a cation exchange chromatography column that had been pre-equilibrated with the equilibrium solution. After loading, unadsorbed protein is washed with equilibration solution, and the absorbance of the washing solution at 280 nm is detected until the absorbance no longer decreases; the solution is then eluted with eluent, and the absorbance of the eluent at 280 nm is detected until the absorbance no longer decreases, and the elution peak is collected, which is intermediate III; the ligands of the cation exchange chromatography column packing are carboxymethyl, sulfonic acid, phosphate, phosphite, and phenol; the eluent is a 0.1 M NaAc and 0.1 M NaCl solution, pH 4.5-5.0; (4) Intermediate III is freeze-dried to obtain the finished hyaluronidase.

2. the preparation technology of hyaluronidase according to claim 1, is characterized in that, In step (2), the balancing solution is a 0.05M sodium acetate solution containing 1.2M (NH4)2SO4, with a pH of 6.0; the eluent is a 0.05M sodium acetate solution containing 0.35-0.4M (NH4)2SO4, with a pH of 6.

0.

3. the preparation technology of hyaluronidase according to claim 2, is characterized in that, In the step (3), the equilibrium solution is 0.1 M NaAc, pH 5.

5.

4. the preparation technology of hyaluronidase according to claim 3, is characterized in that, The preparation process further comprises ultrafiltration of the intermediate III obtained in step (3) to exchange salt, wherein the solution used for ultrafiltration is 0.01M sodium acetate buffer solution with a pH of 6.0-7.0.

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