Purification method of vilasidase beta and liquid medicine

Abstract

The invention discloses a purification method of vilasidase beta and a liquid medicine, and relates to the technical field of protein purification. The purification method of the vilasidase beta comprises the following steps: hydrophobic charge-induced chromatography treatment, anion exchange chromatography treatment, virus inactivation treatment, cation exchange chromatography treatment, hydrophobic chromatography treatment, primary ultrafiltration treatment, virus removal filtration treatment and secondary ultrafiltration treatment, so as to obtain the purified vilasidase beta. The vilasidase beta obtained by the purification method is high in recovery rate and stable in enzyme activity.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular to a purification method and a medicinal solution of velaglucerase beta. Background Art

[0002] Velaglucerase β is a recombinant human glucocerebrosidase with a high mannose exposure at its terminal end. It enters cells through mannose receptor-mediated endocytosis and catalyzes the hydrolysis of glucocerebroside, thereby achieving the purpose of treating Gaucher disease.

[0003] The current method for purifying glucocerebrosidase includes conventional cation exchange chromatography capture, low pH virus inactivation, etc. However, the purity, specific activity and enzyme activity recovery rate of glucocerebrosidase obtained by the existing purification process are poor. Summary of the invention

[0004] The main purpose of the present invention is to provide a purification method and a liquid medicine for velaglucerase β, aiming to solve the problems of low recovery rate and poor enzyme activity stability of the purified velaglucerase β in the prior art.

[0005] To achieve the above object, the present invention provides a method for purifying velaglucerase β, comprising the following steps:

[0006] S10, subjecting the cell culture fluid collected containing velaglucerase β to hydrophobic charge induction chromatography to obtain a first eluate containing velaglucerase β;

[0007] S20, performing anion exchange chromatography on the first eluate to obtain a second eluate containing velaglucerase β;

[0008] S30, performing virus inactivation treatment on the second eluate, and performing cation exchange chromatography on the second eluate after the virus inactivation treatment to obtain a third eluate containing velaglucerase β;

[0009] S40, performing hydrophobic chromatography on the third eluate to obtain a fourth eluate containing velaglucerase β;

[0010] S50, performing a first ultrafiltration treatment on the fourth eluate, replacing the velaglucerase β in the fourth eluate with a virus removal filtration buffer, and sequentially performing a virus removal filtration treatment and a second ultrafiltration treatment on the virus removal filtration buffer to obtain purified velaglucerase β.

[0011] In one embodiment, the method for obtaining the cell culture collection fluid comprises the following steps:

[0012] Cells containing velaglucerase β are obtained, and the cells are subjected to cell recovery, cell expansion and cell perfusion culture to obtain a cell culture collection fluid containing velaglucerase β.

[0013] In one embodiment, in step S10, the filler treated with hydrophobic charge induction chromatography comprises MEP HyperCel filler.

[0014] In one embodiment, step S10 includes:

[0015] The cell culture collection fluid containing velaglucerase β was subjected to hydrophobic charge induction chromatography, and eluted in sequence with 20 mM phosphate buffer at pH 7.2, 50 mM sodium acetate-acetic acid containing 0.5 M arginine hydrochloride at pH 6.0, 50 mM sodium acetate-acetic acid at pH 5.8, and 50 mM sodium acetate-acetic acid at pH 4.8 to obtain a first eluate containing velaglucerase B.

[0016] In one embodiment, performing virus inactivation treatment on the second eluate comprises:

[0017] The pH of the second eluate is adjusted to 5.3-5.7, a detergent is added to the system so that the mass percentage of the detergent is 0.5-1.5%, and the reaction is carried out to obtain a second eluate after virus inactivation treatment;

[0018] Wherein, the detergent includes TritonX-100.

[0019] In one embodiment, the reaction temperature is 18-26°C; and / or,

[0020] The reaction time is ≥60 min.

[0021] In one embodiment, in step S50, the ultrafiltration treatment includes diafiltration; and / or,

[0022] The temperature of the ultrafiltration treatment is 18-26°C.

[0023] The present invention also provides a medicinal solution, comprising velaglucerase β purified by the above-mentioned velaglucerase β purification method.

[0024] In one embodiment, an additive is further included, wherein the mass concentration of the additive in the drug solution is 0.01-0.05%, and the additive includes polysorbate 80.

[0025] In one embodiment, the mass concentration of velaglucerase β in the drug solution is 3.4-4.6 g / L.

[0026] In the technical scheme of the present invention, the velaglucerase β in the cell culture collection fluid containing velaglucerase β is captured by hydrophobic charge induction chromatography treatment, thereby improving the purity, specific activity and enzyme activity recovery rate of velaglucerase β; the four-step chromatography process of hydrophobic charge induction chromatography treatment, anion exchange chromatography treatment, cation exchange chromatography treatment and hydrophobic chromatography treatment makes the finally obtained velaglucerase β have less impurity content, higher purity, higher recovery rate and more stable enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0028] Figure 1 (a) is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) image of the protein component of the eluate during the cation exchange chromatography process in step S10 of Comparative Example 1 provided by the present invention;

[0029] Figure 1 (b) is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) image of the protein component of the eluate during the hydrophobic charge induction chromatography process in step S10 of Comparative Example 1 provided by the present invention.

[0030] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0031] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0032] Velaglucerase β is a recombinant human glucocerebrosidase with a high mannose exposure at its terminal end. It enters cells through mannose receptor-mediated endocytosis and catalyzes the hydrolysis of glucocerebroside, thereby achieving the purpose of treating Gaucher disease.

[0033] The current method for purifying velaglucerase β includes conventional cation exchange chromatography capture, low pH virus inactivation and other steps. However, the purity, specific activity and enzyme activity recovery rate of velaglucerase β obtained by the existing purification process are poor.

[0034] In view of this, the present invention provides a method for purifying velaglucerase β, comprising the following steps:

[0035] S10, subjecting the cell culture fluid collected containing velaglucerase β to hydrophobic charge induction chromatography to obtain a first eluate containing velaglucerase β;

[0036] S20, performing anion exchange chromatography on the first eluate to obtain a second eluate containing velaglucerase β;

[0037] S30, performing virus inactivation treatment on the second eluate, and performing cation exchange chromatography on the second eluate after the virus inactivation treatment to obtain a third eluate containing velaglucerase β;

[0038] S40, performing hydrophobic chromatography on the third eluate to obtain a fourth eluate containing velaglucerase β;

[0039] S50, performing a first ultrafiltration treatment on the fourth eluate, replacing the velaglucerase β in the fourth eluate with a virus removal filtration buffer, and sequentially performing a virus removal filtration treatment and a second ultrafiltration treatment on the virus removal filtration buffer to obtain purified velaglucerase β.

[0040] In the technical scheme of the present invention, since there is no fusion tag that can be used for affinity chromatography in the molecular structure of velaglucerase β, the present invention utilizes its surface charge and hydrophilicity for capture, that is, the velaglucerase β in the cell culture collection fluid containing velaglucerase β is captured by hydrophobic charge induction chromatography treatment, thereby improving the purity, specific activity and enzyme activity recovery rate of velaglucerase β; the four-step chromatography process of hydrophobic charge induction chromatography treatment, anion exchange chromatography treatment, cation exchange chromatography treatment and hydrophobic chromatography treatment makes the finally obtained velaglucerase β have less impurity content, higher purity, higher recovery rate and more stable enzyme activity.

[0041] Specifically, in step S10, compared with other chromatographic methods, hydrophobic charge induction chromatography can more effectively capture velaglucerase β, obtain velaglucerase B with higher purity, specific activity and enzyme activity recovery rate, and its elution volume is also small, the reagent cost is low, and the operation is simple, and there is no need to perform complex pre-treatment on the cell culture harvesting liquid. In step S20, anion exchange chromatography is used to remove impurities such as residual cell proteins and DNA and to preliminarily remove viruses. In step S30, the virus inactivation treatment is used to further process the virus, and the second eluate after the virus inactivation treatment is subjected to cation exchange chromatography treatment to further purify velaglucerase β, remove process-related impurities such as cell proteins and product-related impurities such as polymers and fragments. In step S40, the hydrophobic chromatography treatment can further purify the protein by the hydrophobic chromatography filler ligand and the hydrophobic surface interaction of velaglucerase β. In step S50, the second ultrafiltration treatment is used to replace the velaglucerase β in the virus removal filter buffer after the virus removal filter treatment into the buffer of velaglucerase β.

[0042] In some embodiments of the present invention, the method for obtaining the cell culture collection fluid comprises the following steps: obtaining cells containing velaglucerase β, performing cell recovery, cell expansion and cell perfusion culture on the cells, and obtaining a cell culture collection fluid containing velaglucerase β. Among them, obtaining cells containing velaglucerase β means obtaining a cell line capable of expressing velaglucerase β. These cells may be genetically engineered to ensure that they can efficiently produce velaglucerase β; the process of restoring cryopreserved cells to an active growth state through cell recovery; more cells containing velaglucerase β can be obtained through cell expansion for subsequent protein production; cell perfusion culture is a continuous cell culture technology, in which fresh culture medium is continuously added to the cells, while removing metabolic waste and other unnecessary components, which helps to maintain the cells in a good growth state and increase the yield of velaglucerase β. After the above steps, what is finally obtained is a cell culture fluid rich in velaglucerase β.

[0043] In some embodiments of the present invention, in step S10, the filler treated with hydrophobic charge induction chromatography includes MEP HyperCel filler. Compared with other fillers, MEP HyperCel filler has high loading capacity and can adsorb more velaglucerase β, thereby improving the purification efficiency of velaglucerase β.

[0044] In some embodiments of the present invention, step S10 includes:

[0045] The cell culture collection fluid containing velaglucerase β was subjected to hydrophobic charge induction chromatography, and eluted with 20mM phosphate buffer at pH 7.2, 50mM sodium acetate-acetic acid containing 0.5M arginine hydrochloride at pH 6.0, 50mM sodium acetate-acetic acid at pH 5.8, and 50mM sodium acetate-acetic acid at pH 4.8, to obtain the first eluate containing velaglucerase β. Among them, the phosphate buffer is used to balance the chromatography column, so that the column is in the best state and ready to receive the sample; sodium acetate-acetic acid buffers with different pH values ​​are used for elution in turn, and proteins with different binding strengths are gradually eluted, which can effectively remove most non-target proteins, thereby improving the purity and recovery rate of velaglucerase β. At the same time, the sodium acetate-acetic acid buffer system can protect the enzyme activity of velaglucerase β and make it more stable during the elution process.

[0046] In some embodiments of the present invention, performing virus inactivation treatment on the second eluate comprises:

[0047] The pH of the second eluate is adjusted to 5.3-5.7, a detergent is added to the system so that the mass percentage of the detergent is 0.5-1.5%, and the reaction is carried out to obtain a second eluate after virus inactivation treatment;

[0048] Wherein, the detergent includes TritonX-100.

[0049] In the technical solution of the present invention, the use of a detergent can effectively destroy the viral envelope and inactivate the virus. Compared with conventional low pH inactivation, the use of a detergent for virus inactivation can better maintain the enzymatic activity of velaglucerase β. Triton X-100, also known as polyoxyethylene octylphenyl ether, is milder than other detergents and is conducive to maintaining the enzymatic activity of velaglucerase β.

[0050] In some embodiments of the present invention, the reaction temperature is 18-26°C; and / or the reaction time is ≥60 min. The reaction temperature can be 18°C, 22°C or 26°C; the reaction time can be 60 min, 2 h, 6 h or 24 h, or even longer. The reaction temperature and time within the above range can ensure a good virus inactivation effect.

[0051] In some embodiments of the present invention, in step S50, the ultrafiltration treatment includes diafiltration; and / or the temperature of the ultrafiltration treatment is 18-26° C. The ultrafiltration treatment can displace velaglucerase beta from one solution to another solution.

[0052] The present invention also provides a liquid medicine, comprising velaglucerase β purified by the above-mentioned method for purifying velaglucerase β, and thus having all the beneficial effects of the above-mentioned method for purifying velaglucerase β, which will not be described in detail here.

[0053] In some embodiments of the present invention, an additive is further included, and the mass concentration of the additive in the drug solution is 0.01-0.05%, and the additive includes polysorbate 80. The mass concentration of the additive in the drug solution can be 0.01%, 0.03% or 0.05%, and its mass concentration within the above range can ensure good stability of velaglucerase β.

[0054] In some embodiments of the present invention, the mass concentration of the velaglucerase beta in the liquid medicine is 3.4-4.6 g / L. The mass concentration of the velaglucerase beta in the liquid medicine can be 3.4 g / L, 4 g / L or 4.6 g / L, and its mass concentration within the above range can simultaneously ensure that the liquid medicine has a good effect and a low cost.

[0055] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0056] Example 1

[0057] This embodiment provides a method for purifying velaglucerase β, comprising the following steps:

[0058] S10. Hydrophobic charge induction chromatography: The cell culture collection fluid containing velaglucerase β was subjected to hydrophobic charge induction chromatography using MEP HyperCel as filler, and eluted with 20 mM phosphate buffer at pH 7.2, 50 mM sodium acetate-acetic acid containing 0.5 M arginine hydrochloride at pH 6.0, 50 mM sodium acetate-acetic acid at pH 5.8, and 50 mM sodium acetate-acetic acid at pH 4.8, and 0.025 AU / mm-elution peak-0.025 AU / mm was collected to obtain the first eluate containing velaglucerase B.

[0059] S20, anion exchange chromatography treatment: anion exchange chromatography is performed using conventional commercially available anion fillers, Tris-acetate buffer is used as a balancing and post-sampling elution buffer, and the second eluate is collected.

[0060] S30, virus inactivation treatment: at 18°C, add Triton X-100 to a final mass percentage of 0.5%, incubate for 60 minutes, and obtain a virus inactivated sample; then, perform cation exchange chromatography on the virus inactivated sample, using conventional commercially available cation exchange fillers, and washing and eluting with acetic acid-sodium acetate buffer to obtain a third eluate.

[0061] S40, hydrophobic chromatography treatment: using conventional commercially available hydrophobic chromatography filler, eluting with acetic acid-sodium acetate buffer containing ammonium sulfate, eluting with arginine hydrochloric acid buffer, and collecting the fourth eluate;

[0062] S50, first ultrafiltration treatment: using Merck's Pellicon 3 series ultrafiltration membrane package for concentration and buffer exchange, adjusting the protein concentration to 1 g / L after diafiltration;

[0063] Virus removal filtration treatment: Asahi Kasei virus filter was used and balanced with sodium citrate buffer. During the filtration process, the virus was retained and velaglucerase β passed through the filter into the collection container;

[0064] The second ultrafiltration treatment was performed by using Merck's Pellicon 3 series ultrafiltration membrane package for concentration and buffer exchange, and the sample concentration was adjusted to 4.0 g / L with a buffer containing sodium citrate and sucrose to obtain purified velaglucerase β.

[0065] Example 2

[0066] Compared with Example 1, Example 2 is different in that:

[0067] In step S30, the mass percentage of Triton X-100 is 0.5%, and the reaction time is 24 h.

[0068] Example 3

[0069] Compared with Example 1, Example 3 is different in that:

[0070] In step S30, the mass percentage of Triton X-100 is 1.0%, and the reaction time is 6 h.

[0071] Example 4

[0072] Compared with Example 1, Example 4 is different in that:

[0073] In step S30, the mass percentage of Triton X-100 is 1.0%, and the reaction time is 24 h.

[0074] Example 5

[0075] Compared with Example 1, Example 5 is different in that:

[0076] In step S30, the mass percentage of Triton X-100 is 1.5%, and the reaction time is 6 hours.

[0077] Example 6

[0078] Compared with Example 1, Example 6 is different in that:

[0079] In step S30, the mass percentage of Triton X-100 is 1.5%, and the reaction time is 24 hours.

[0080] Example 7

[0081] The present embodiment provides a medicinal solution, comprising the purified velaglucerase β prepared in Example 1, polysorbate 80 and a diluent; in the medicinal solution, the mass concentration of the purified velaglucerase β prepared in Example 1 is 4 g / L, and the mass concentration of polysorbate 80 is 0.03 g / L; in the diluent, the molar concentration of sodium citrate is 20 mM, the concentration of sucrose is 8% (w / v), and the pH is 6.0.

[0082] Comparative Example 1

[0083] Compared with Example 1, Comparative Example 1 is different in that:

[0084] In step S10, the hydrophobic charge induction chromatography treatment is replaced by a cation exchange chromatography treatment, using Thermo's filler POROS XS (Cat. No. 440443), 50 mM acetic acid-sodium acetate buffer for balancing and elution, and 50 mM acetic acid-sodium acetate buffer containing 1 M sodium chloride solution for elution.

[0085] Comparative Example 2

[0086] Compared with Example 1, Comparative Example 2 is different in that:

[0087] In step S30, the virus inactivation method is low pH inactivation, the pH value is 3.80, and the reaction time is 2 hours at 18°C.

[0088] Comparative Example 3

[0089] Compared with Example 1, Comparative Example 3 is different in that:

[0090] In step S10, the hydrophobic charge induction chromatography treatment is replaced by a cation exchange chromatography treatment, the filler is POROSXS, 50 mM acetic acid-sodium acetate buffer is used for balancing and elution, and 50 mM acetic acid-sodium acetate buffer containing 1 M sodium chloride solution is used for elution.

[0091] In step S30, the virus inactivation method is low pH inactivation, the pH value is 3.80, and the reaction time is 2 hours at 18°C.

[0092] Comparative Example 4

[0093] Compared with Example 2, Comparative Example 4 is different in that:

[0094] No Triton X-100 was added, the pH value was adjusted to 3.80, and the reaction time was 2 h.

[0095] Comparative Example 5

[0096] Compared with Example 1, Comparative Example 6 is different in that:

[0097] Triton X-100 was not added and the reaction time was 24 h.

[0098] Comparative Example 6

[0099] Compared with Example 2, Comparative Example 7 is different in that:

[0100] No Triton X-100 was added, the pH value was adjusted to 3.70, and the reaction time was 2 h.

[0101] Comparative Example 7

[0102] Compared with Example 2, Comparative Example 8 is different in that:

[0103] No Triton X-100 was added, the pH value was adjusted to 3.60, and the reaction time was 2 h.

[0104] Performance Testing

[0105] The protein components of the eluate in the chromatography process of step S10 in Example 1 and Comparative Example 1 were analyzed by SDS-PAGE. The results are as follows: Figure 1 a and Figure 1 As shown in b.

[0106] Figure 1 In a: M is the protein molecular weight standard, L is the loading sample, FT is the flow-through sample, W1 is the elution 1 sample, W2 is the elution 2 sample, and E is the elution sample; Figure 1 In b: M is the protein molecular weight standard, L is the loading sample, L5.5 is the loading sample adjusted to pH 5.5, FT is the flow-through sample, and E1-E5 are different components of the elution peak.

[0107] The molecular weight of velaglucerase beta is 55.6 kDa. Figure 1 a and Figure 1 b It can be seen that the eluted samples of cation exchange chromatography and hydrophobic charge induction chromatography both contain the target protein velaglucerase β band, but the hydrophobic charge induction chromatography band is wider and more obvious, indicating that step S10 in Example 1 has a higher enzyme activity recovery rate.

[0108] The specific activity and enzyme activity recovery rate of velaglucerase β in the first eluate after chromatography in step S10 in Example 1 and Comparative Example 1 were further detected, wherein the specific activity was detected by ELISA method, and the enzyme activity recovery rate was obtained by the ratio of the total enzyme activity of the eluted sample to the total enzyme activity of the loaded sample. The results are shown in Table 1.

[0109] Table 1 Characterization of velaglucerase β in the first eluate of Example 1 and Comparative Example 1

[0110] Specific activity (U / mg) Enzyme activity recovery rate (%) Example 1 19.2 114.4 Comparative Example 1 8.5 68.1

[0111] As shown in Table 1, compared with cation exchange chromatography, hydrophobic charge induction chromatography has higher specific activity and enzyme activity recovery rate of velaglucerase β.

[0112] The specific activity of velaglucerase β after the virus inactivation treatment in step S30 in Examples 1 to 6 and Comparative Example 5 was detected, with the reaction time of 0 h as the control group. The results are shown in Table 2.

[0113] Table 2 Specific activity of velaglucerase β after virus inactivation treatment in Examples 1 to 6 and Comparative Example 5

[0114] Specific activity (U / mg) Control group 11.6 Example 1 12.5 Example 2 9.5 Example 3 13.2 Example 4 10.4 Example 5 13.3 Example 6 10.5 Comparative Example 5 8.7

[0115] As shown in Table 2, the use of Triton X100 detergent, with a concentration of 0.5-1.5%, and a reaction time of 6-24 hours, had no significant effect on the comparative activity. Therefore, the detergent virus inactivation treatment under this condition is conducive to the stabilization of enzyme activity.

[0116] The specific activity of velaglucerase β after the virus inactivation treatment in step S30 in Comparative Examples 4 and Comparative Examples 6-7 was detected, and the group without pH adjustment was used as the blank group. The results are shown in Table 3.

[0117] Table 3 Specific activity of velaglucerase β after virus inactivation treatment in Comparative Examples 4 and 6-7

[0118]

[0119] As shown in Table 3, the specific activity of velaglucerase β decreased by more than 50% after incubation for 2 h at pH 3.80 or below.

[0120] By comparing Table 2 and Table 3, it can be seen that velaglucerase β loses its activity significantly under low pH conditions, while the detergent inactivation method used in the present invention is milder to the enzyme, and the specific activity does not change significantly before and after the treatment. This indicates that the virus inactivation treatment method of the present invention is more suitable for maintaining the enzyme activity stability of velaglucerase β.

[0121] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for purifying velaglucerase β, characterized in that: The following steps are involved: S10, subjecting the cell culture fluid collected containing velaglucerase β to hydrophobic charge induction chromatography to obtain a first eluate containing velaglucerase β; S20, performing anion exchange chromatography on the first eluate to obtain a second eluate containing velaglucerase β; S30, performing virus inactivation treatment on the second eluate, and performing cation exchange chromatography on the second eluate after the virus inactivation treatment to obtain a third eluate containing velaglucerase β; S40, performing hydrophobic chromatography on the third eluate to obtain a fourth eluate containing velaglucerase β; S50, performing a first ultrafiltration treatment on the fourth eluate, replacing the velaglucerase β in the fourth eluate with a virus removal filtration buffer, and sequentially performing a virus removal filtration treatment and a second ultrafiltration treatment on the virus removal filtration buffer to obtain purified velaglucerase β.

2. The method for purifying velaglucerase β according to claim 1, characterized in that: In step S10, the method for obtaining the cell culture collection fluid comprises the following steps: Cells containing velaglucerase β are obtained, and the cells are subjected to cell recovery, cell expansion and cell perfusion culture to obtain a cell culture collection fluid containing velaglucerase β.

3. The method for purifying velaglucerase β according to claim 1, characterized in that: In step S10, the filler treated with hydrophobic charge induction chromatography includes MEP HyperCel filler.

4. The method for purifying velaglucerase β according to claim 3, characterized in that: Step S10 includes: The cell culture collection fluid containing velaglucerase β was subjected to hydrophobic charge induction chromatography, and eluted in sequence with 20 mM phosphate buffer at pH 7.2, 50 mM sodium acetate-acetic acid containing 0.5 M arginine hydrochloride at pH 6.0, 50 mM sodium acetate-acetic acid at pH 5.8, and 50 mM sodium acetate-acetic acid at pH 4.8 to obtain a first eluate containing velaglucerase β.

5. The method for purifying velaglucerase β according to claim 1, characterized in that: In step S30, performing virus inactivation treatment on the second eluate includes: The pH of the second eluate is adjusted to 5.3-5.7, a detergent is added to the system so that the mass percentage of the detergent is 0.5-1.5%, and the reaction is carried out to obtain a second eluate after virus inactivation treatment; Wherein, the detergent includes TritonX-100.

6. The method for purifying velaglucerase β according to claim 5, characterized in that: The reaction temperature is 18-26°C; and / or, The reaction time is ≥60 min.

7. The method for purifying velaglucerase β according to claim 1, characterized in that: In step S50, the ultrafiltration treatment includes diafiltration; and / or, The temperature of the ultrafiltration treatment is 18-26°C.

8. A liquid medicine, characterized in that: The invention comprises the velaglucerase β purified by the velaglucerase β purification method according to any one of claims 1 to 7.

9. The liquid medicine according to claim 8, characterized in that The invention also comprises an additive, wherein the mass concentration of the additive in the drug solution is 0.01-0.05%, and the additive comprises polysorbate 80.

10. The liquid medicine according to claim 8, characterized in that The mass concentration of the velaglucerase β in the drug solution is 3.4 to 4.6 g / L.

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