Isolation and purification method of anti-pcsk9 monoclonal antibody

Abstract

The application belongs to the technical field of chemical medicines and relates to a separation and purification method of an anti-PCSK9 monoclonal antibody. After the cell culture solution of the antibody A is filtered, the crude solution of the antibody A is obtained. Then, after being sequentially subjected to affinity chromatography, hydrophobic chromatography, reverse phase chromatography and cation chromatography, impurities in the antibody A can be removed, the requirement of a pharmacopoeia is met, and the antibody A with high purity and high yield is obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a separation and purification method of an anti-PCSK9 monoclonal antibody. BACKGROUND

[0002] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a proprotein convertase belonging to the subtilisin / kexin family of secreted proteases. Evidence shows that PCSK9 promotes the degradation of low-density lipoprotein receptor (LDLR) and thus increases the content of LDL cholesterol in plasma, while LDLR mediates the endocytosis of LDL in the liver, which is the main pathway for removing LDL from the circulatory system.

[0003] The anti-PCSK9 monoclonal antibody is a full human monoclonal antibody, belongs to an IgG4 subtype antibody, contains two complete light chains and heavy chains, and has a molecular weight of about 148.3 KD. The antibody can selectively bind to PCSK9, block the interaction between PCSK9 and LDLR, increase the circulation of LDLR, and thus increase the level of LDLR on the surface of hepatocytes. A high content of LDLR can bind to LDL in plasma and then transport the LDL to lysosomes for degradation, so as to reduce the level of LDL and also reduce the levels of other lipoproteins.

[0004] High-purity anti-PCSK9 monoclonal antibodies can reduce adverse reactions and improve storage stability. The anti-PCSK9 monoclonal antibody CHO cell culture solution contains many impurities such as host proteins and cytochromes, and has low purity. Therefore, it is of high application significance to develop a purification process of high-purity anti-PCSK9 monoclonal antibodies. SUMMARY

[0005] In view of the problems in the prior art, the application provides a separation and purification method of an anti-PCSK9 monoclonal antibody, which can obtain high-purity anti-PCSK9 monoclonal antibodies.

[0006] The application provides a separation and purification method of an anti-PCSK9 monoclonal antibody, which specifically comprises the following steps:

[0007] A separation and purification method of a PCSK9 monoclonal antibody, characterized by comprising the following steps:

[0008] (1) Filtration, filtering the cell culture solution of the antibody A to obtain a crude solution of the antibody A;

[0009] (2) Affinity chromatography, subjecting the crude solution of the antibody A to affinity chromatography to obtain a clear solution of the antibody A;

[0010] (3) Hydrophobic chromatography, subjecting the antibody A treated in step (2) to hydrophobic chromatography;

[0011] (4) reverse phase chromatography; the antibody A treated in step (3) is treated by reverse phase chromatography; and

[0012] (5) cationic chromatography; the antibody A treated in step (4) is treated by cationic chromatography to remove impurities in the antibody A, so as to obtain the antibody A with high purity.

[0013] As a preferred technical solution of the present application, the antibody A is selected from antibody 18.156.8, Evolocumab, Alirocumab, recaticimab or tafolecimab.

[0014] As a preferred technical solution of the present application, the affinity chromatography comprises the following steps:

[0015] a. Equilibrium buffer is used to equilibrate the affinity chromatography column filled with filler;

[0016] b. Sample loading: the crude liquid of antibody A obtained in step (1) is loaded into the chromatography column equilibrated in step a, the loading capacity is less than or equal to 50 mg / ml, the sample retention time is 9-27 min, and the unbound substances are removed by washing with the equilibrium buffer in step a;

[0017] c. Elution: the target protein is eluted with elution collection buffer;

[0018] d. The target protein treated in step c is collected, and the pH of the target protein is adjusted by using the collection liquid.

[0019] As a preferred technical solution of the present application, the equilibrium buffer in step a is selected from at least one of PBS, MES, NaAC, preferably PBS.

[0020] As a preferred technical solution of the present application, the concentration of the equilibrium buffer in step a is 30 mmol / L-80 mmol / L, pH 6.0-7.0, preferably 50 mmol / L, 60 mmol / L and 70 mmol / L, pH 6.5±0.2.

[0021] As a preferred technical solution of the present application, the elution collection buffer in step c is selected from at least one of Gly-HCL and NaAC, preferably Gly-HCL.

[0022] As a preferred technical solution of the present application, the concentration of the elution buffer in step c is 40 mmol / L-70 mmol / L, pH 2.5-4.5, preferably 50 mmol / L, 60 mmol / L, pH 3.0±0.2.

[0023] As a preferred technical solution of the present application, the collection liquid in step d is selected from at least one of Tris-HCL, PBS, MES, NaAC, preferably Tris-HCL and PBS. For example, the pH of the target protein is adjusted to 6.5±0.2 using Tris-HCL or PBS.

[0024] As a preferred technical solution of the present application, the concentration of the collection liquid in step d is selected from 0.1-3mmol / L, preferably 1mmol / L and 1.5mmol / L, and the pH is 9.0±0.2.

[0025] As a preferred technical solution of the present application, the hydrophobic chromatography comprises the following steps:

[0026] a. Equilibrium: equilibrate the hydrophobic chromatography filler with an equilibrium buffer;

[0027] b. Sample loading: load the antibody A sample prepared in the above step (2) into the hydrophobic chromatography column equilibrated in step a, wherein the sample loading capacity is 10-60mg / ml, the sample retention time is 9-27min, the unbound material is removed by washing with the equilibrium buffer of step a, and the linear elution flow rate is 100cm / h-300cm / h.

[0028] As a preferred technical solution of the present application, the equilibrium buffer of step a is selected from at least one of Tris-HCL, PBS, NaAC, preferably Tris-HCL.

[0029] As a preferred technical solution of the present application, the concentration of the equilibrium buffer of step a is selected from 10mmol / L-70mmol / L, preferably 30mmol / L, and the pH is 6.0-8.0, preferably pH 6.5±0.2.

[0030] As a preferred technical solution of the present application, the reverse phase chromatography comprises the following steps:

[0031] a. Equilibrium: equilibrate the reverse phase chromatography filler with an equilibrium buffer;

[0032] b. Sample loading: load the antibody A sample prepared in the above step (3) into the reverse phase chromatography column equilibrated in step a, wherein the sample loading capacity is 5-30mg / ml, the sample retention time is 9-18min, the unbound material is removed by washing with the equilibrium buffer of step a, and the linear elution flow rate is 100cm / h-200cm / h;

[0033] c. Elution: elute the target protein antibody A with an elution buffer.

[0034] As a preferred technical solution of the present application, the equilibrium buffer in the reversed-phase chromatography is selected from acetonitrile, and the acetonitrile concentration is selected from 10%-13%; the elution buffer is selected from acetonitrile, and the acetonitrile concentration is selected from 25%-30%.

[0035] As a preferred technical solution of the present application, the cationic chromatography comprises the following steps:

[0036] a. Equilibrium: using the equilibrium buffer to equilibrate the cationic chromatography filler;

[0037] b. Sample loading: loading the antibody A in step (4) above, the sample loading capacity is 10-50 mg / ml, ensuring that the sample is in contact with the filler for 9-27 min, and using the equilibrium buffer in step a to flush to remove unbound substances;

[0038] c. Elution: using the elution buffer to elute the antibody A treated in step b.

[0039] As a preferred technical solution of the present application, the equilibrium buffer in step a is selected from at least one of sodium chloride and Tris-HCL, NaAC, PBS, and MES, preferably sodium chloride and Tris-HCL, and PBS.

[0040] As a preferred technical solution of the present application, the equilibrium buffer in step a has a concentration of 10 mmol / L-100 mmol / L, preferably 50 mmol / L and 60 mmol / L; the concentration of sodium chloride is 0.2-0.5 mol / L, preferably 0.3 mol / L, and the pH is 5.0-8.0, preferably pH 6.5±0.2.

[0041] As a preferred technical solution of the present application, the filler of the affinity chromatography is selected from AT ProteinA Diamond plus, NMab TM ProteinA, or MabSelect, preferably AT ProteinA Diamond plus; the filler of the hydrophobic chromatography is selected from Diamond Phenyl(HS), Capto Phenyl, or Uni HR Phenyl-80L, preferably Diamond Phenyl(HS); the filler of the reversed-phase chromatography is selected from Best Poly 15RPC, Source 15RPC, or UniPS, preferably Best Poly 15RPC; and the filler of the cationic chromatography is selected from UniGel-80SP, SP Sepharose FF, or SPBestarose FF, preferably UniGel-80SP.

[0042] The present application has the following advantages and beneficial effects relative to the prior art:

[0043] 1. The application provides a method for separating and purifying anti-PCSK9 monoclonal antibodies, which comprises affinity chromatography, hydrophobic chromatography, reverse phase chromatography and cationic chromatography, so that the target protein with high purity and high yield can be obtained, and the residual amount of acetonitrile and impurities can be controlled within a safe range.

[0044] 2. The application provides a method for separating and purifying anti-PCSK9 monoclonal antibodies, which comprises screening of each chromatography buffer and eluent, so that the target protein with high total yield and good biological activity can be obtained.

[0045] 3. The application provides a method for separating and purifying anti-PCSK9 monoclonal antibodies, which has strong applicability, good repeatability and stability, and can be used in large-scale purification process of anti-PCSK9 monoclonal antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 Screening results of affinity chromatography buffer;

[0047] Figure 2 Screening results of affinity chromatography buffer concentration;

[0048] Figure 3 Screening results of affinity chromatography buffer pH;

[0049] Figure 4 Screening results of affinity chromatography elution collection buffer pH;

[0050] Figure 5 Screening results of affinity chromatography elution collection buffer pH;

[0051] Figure 6 Screening results of affinity chromatography elution collection buffer pH;

[0052] Figure 7 Screening results of hydrophobic chromatography buffer;

[0053] Figure 8 Screening results of hydrophobic chromatography buffer concentration;

[0054] Figure 9 Screening results of hydrophobic chromatography buffer pH;

[0055] Figure 10 Screening results of reverse phase chromatography buffer concentration;

[0056] Figure 11 Screening results of reverse phase chromatography elution buffer concentration;

[0057] Figure 12Results of the purification method described in Example 12 were used.

[0058] Figure 13 Antibody purity and acetonitrile residual amount of the purification method described in Example 12 were used.

[0059] Figure 14 Antibody purity and acetonitrile residual amount of the purification method described in Comparative Example 1 were used.

[0060] Figure 15 Results of the purification method described in Comparative Example 2 were used. DETAILED DESCRIPTION

[0061] The present application will be further described in conjunction with the following examples, but the embodiments of the present application are not limited thereto.

[0062] In the following examples, the preparation of the anti-PCSK9 monoclonal antibody (hereinafter referred to as antibody A) used the method described in patent document CN201710816808.0, or all the contents of this patent document are incorporated by reference into the present application. Antibody A is selected from the sequence corresponding to antibody 18.156.8 in patent document CN201710816808.0.

[0063] Example 1 Screening of affinity chromatography equilibration buffer

[0064] This example illustrates the effect of different types of step a affinity chromatography equilibration buffer on the yield of pure antibody A. Under the same conditions of other purification (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic loading of less than 50 mg / ml, equilibration buffer concentration of 50 mM, pH 6.5, elution buffer selected as 50 mM Gly-HCL, pH 3.0), the equilibration buffer types were selected as Tris-HCL, PBS, MES, and NaAC. The antibody A purification yield results are shown in Table 1. Figure 1 The results show that when the equilibration buffer is PBS, MES, and NaAC, the antibody A purification yield is higher, and PBS is preferred.

[0065] Example 2 Screening of affinity chromatography equilibration buffer concentration

[0066] This example illustrates the effect of different concentrations of the equilibration buffer in step a affinity chromatography on the yield of purified antibody A. In the same conditions of other purification (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of less than 50 mg / ml, equilibration buffer selected as PBS, pH 6.5, elution buffer selected as 50 mM Gly-HCL, pH 3.0), the equilibration buffer concentration was selected as 10 mM, 30 mM, 50 mM, 70 mM, and 100 mM, respectively. The results of the yield of purified antibody A are shown in Table 1. Figure 2 The results show that when the equilibration buffer concentration is 30-70 mM, the yield of purified antibody A is higher, and 50 mM is preferred.

[0067] Example 3: Screening of pH of equilibration buffer in affinity chromatography

[0068] This example illustrates the effect of different pH of the pre- and post- equilibration buffer in step a affinity chromatography on the yield of purified antibody A. In the same conditions of other purification (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of less than 50 mg / ml, pre- and post- equilibration buffer selected as PBS, concentration of 50 mM, elution buffer selected as 50 mM Gly-HCL, pH 3.0), the pH of the pre- and post- equilibration buffer was selected as 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The results of the yield of purified antibody A are shown in Table 2. Figure 3 The results show that when the pH of the pre- and post- equilibration buffer is 6.0-7.0, the yield of purified antibody A is higher, and pH 6.5 is preferred.

[0069] Example 4: Screening of pH of elution and collection buffer in affinity chromatography

[0070] This example illustrates the effect of different types of elution and collection buffer in step a affinity chromatography on the yield of purified antibody A. In the same conditions of other purification (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of less than 50 mg / ml, equilibration buffer selected as PBS, concentration of 50 mM, pH 6.5, elution and collection buffer concentration selected as 50 mM, pH selected as 3.0), the type of elution and collection buffer was selected as Gly-HCL, citrate, and NaAC, respectively. The results of the yield of purified antibody A are shown in Table 3. Figure 4 The results show that when the type of elution and collection buffer is selected as Gly-HCL and NaAC, the yield of purified antibody A is higher, and Gly-HCL is preferred.

[0071] Example 5: Screening of concentration of elution and collection buffer in affinity chromatography

[0072] This example illustrates the effect of different concentrations of elution collection buffer on the yield of purified antibody A in step a. The purification conditions were the same (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of less than 50 mg / ml, and equilibration buffer selected as PBS with a concentration of 50 mM and pH of 6.5, and elution buffer selected as NaAC), and the elution collection buffer concentrations were selected as 10 mM, 30 mM, 50 mM, 70 mM, and 100 mM. The antibody A purification yield results are shown in Table 1. Figure 5 The results show that the antibody A purification yield is higher when the elution collection buffer concentration is 40-70 mM, and 50 mM is preferred.

[0073] Example 6: Screening of pH of elution collection buffer in affinity layer chromatography

[0074] This example illustrates the effect of different pH of elution collection buffer on the yield of purified antibody A in step a. The purification conditions were the same (linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of less than 50 mg / ml, and equilibration buffer selected as PBS with a concentration of 50 mM and pH of 6.5, and elution buffer selected as Gly-HCL with a concentration of 50 mM), and the elution collection buffer pH was selected as 2.5, 2.7, 3.0, 3.3, 3.6, 4.0, 4.3, and 4.5. The antibody A purification yield results are shown in Table 2. Figure 6 The results show that the antibody A purification yield is higher when the elution collection buffer pH is 2.5-4.0, and pH of 3.0 is preferred.

[0075] Example 7: Screening of equilibration buffer in hydrophobic layer chromatography

[0076] This example illustrates the effect of different types of equilibration buffer on the yield and purity of purified antibody A in step b. The purification conditions were the same (hydrophobic chromatography steps and parameters are shown in Table 2), and in the hydrophobic chromatography step: linear elution flow rate of 100 cm / h-300 cm / h, column height of 15 cm±5 cm, sample chromatography retention time of 9-27 min, dynamic load of 10-60 mg / ml, equilibration buffer concentration of 30 mM and pH of 6.5, and equilibration buffer type selected as Tris-HCL, PBS, MES, and NaAC. The antibody A purification yield and purity results are shown in Table 3. Figure 7 The results show that the antibody A purification yield and purity are higher when the equilibration buffer is Tris-HCL or PBS, and Tris-HCL is preferred.

[0077] Example 8 Screening of hydrophobic chromatography equilibration buffer concentration

[0078] This example illustrates the effect of different concentrations of equilibration buffer on the yield of purified antibody A in step b hydrophobic chromatography. The other purification conditions were the same (see Table 2 for affinity chromatography parameters), the linear elution flow rate in hydrophobic chromatography was 100 cm / h-300 cm / h, the column height was 15 cm±5 cm, the sample chromatography retention time was 9-27 min, the dynamic loading capacity was 10-60 mg / ml, and the equilibration buffer type was Tris-HCL, pH 6.5, and the equilibration buffer concentration was selected to be 10 mM, 30 mM, 50 mM, 70 mM, and 100 mM. The antibody A purification yield and purity results are shown in Table 3. Figure 8 The results show that when the equilibration buffer concentration is 10-70 mM, the antibody A purification yield and purity are higher, and the equilibration buffer concentration of 30 mM is preferred.

[0079] Example 9 Screening of hydrophobic chromatography equilibration buffer pH

[0080] This example illustrates the effect of different pH of equilibration buffer on the yield and purity of purified antibody A in step b hydrophobic chromatography.

[0081] The other purification conditions were the same (see Table 1 below for affinity chromatography parameters), the hydrophobic chromatography step: linear elution flow rate was 100 cm / h-300 cm / h, column height was 15 cm±5 cm, sample chromatography retention time was 9-27 min, dynamic loading capacity was 10-60 mg / ml, and the equilibration buffer type was Tris-HCL, concentration 30 mM, and the equilibration buffer pH was selected to be 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0. The antibody A purification yield results are shown in Table 4. Figure 9 The results show that when the equilibration buffer pH is 6.0-7.0, the antibody A purification yield and purity are higher, and the equilibration buffer pH of 6.5 is preferred.

[0082] Example 10 Screening of reverse phase chromatography equilibration buffer concentration

[0083] This example illustrates the effect of different concentrations of acetonitrile in equilibration buffer on the yield and purity of purified antibody A in step c reverse phase chromatography.

[0084] Under the same purification conditions (affinity chromatography parameters are shown in Table 1 below, and hydrophobic chromatography parameters are shown in Table 2 below), the reversed-phase chromatography steps were as follows: linear elution flow rate of 100 cm / h-200 cm / h, column height of 15 cm ± 5 cm, sample retention time of 9-18 min, dynamic loading of 5-30 mg / ml, and elution collection buffer of 30% acetonitrile aqueous solution. Equilibration buffers were selected from 5% acetonitrile aqueous solution, 8% acetonitrile aqueous solution, 11% acetonitrile aqueous solution, 13% acetonitrile aqueous solution, and 15% acetonitrile aqueous solution. The antibody A purification yield and purity results are shown below. Figure 10 The results showed that using a 10-15% acetonitrile aqueous solution as the equilibration buffer resulted in higher antibody A purity, while using a 10-13% acetonitrile aqueous solution resulted in higher antibody A yield and purity. The preferred acetonitrile aqueous solution concentration was 13%.

[0085] Example 11 Screening of Reverse Chromatography Elution Buffer Concentration

[0086] This example illustrates the effect of different acetonitrile concentrations in the reverse-phase chromatography elution and collection buffer on the yield and purity of purified antibody A.

[0087] Under the same purification conditions (affinity chromatography parameters are shown in Table 1 below, hydrophobic chromatography parameters are shown in Table 2 below, and cation chromatography parameters are shown in Table 3 below), the reversed-phase chromatography steps were as follows: linear elution flow rate of 100-200 cm / h, column height of 15 cm ± 5 cm, sample retention time of 9-18 min, dynamic loading of 5-30 mg / ml, pre- and post-equilibration buffers of 11% acetonitrile aqueous solution, and elution collection buffers of 20%, 23%, 26%, 28%, and 30% acetonitrile aqueous solutions, respectively. The antibody A purification yield and purity results are shown in [Table 3]. Figure 11 The results showed that using a 25-30% acetonitrile aqueous solution as the elution and collection buffer resulted in a higher antibody A yield, with a preferred acetonitrile aqueous solution concentration of 26%.

[0088] Example 12 Purification method of antibody A

[0089] Step (1): Filtering

[0090] CHO cell culture medium containing antibody A is centrifuged and filtered to obtain crude antibody A solution, which is used as the purification stock solution. Centrifugation and filtration are performed using a 0.22 μm filter membrane under the following conditions: 4000-8000 g, 7-12 min, 2-8℃. Alternatively, CHO cell culture medium containing antibody A can be subjected to deep filtration using a two-stage tandem DOHC and BIHC filter from Millipore, followed by filtration through a 0.2 μm filter cartridge, to obtain crude antibody A solution, which is also used as the purification stock solution.

[0091] Step (2): Affinity chromatography

[0092] The purified stock solution obtained in step (1) above was subjected to crude purification by affinity chromatography. The steps of affinity chromatography are as follows:

[0093] a. Equilibrate the affinity chromatography column after it has been packed with packing material using equilibration buffer;

[0094] b. Loading: Load the purified stock solution obtained in step (1) into the chromatography column after equilibration in step a above. The column height is 15cm ± 5cm, the loading capacity is less than or equal to 50mg / ml, the sample retention time is 9-27min, and the unbound material is washed with the equilibration buffer in step a above.

[0095] c. Elution: Elute the target protein with elution collection buffer;

[0096] d. Collect the target protein after treatment in step c, and adjust the pH of the target protein to 6.5±0.2 using the collection solution.

[0097] The parameters and / or conditions involved in each step of affinity chromatography are shown in Table 1.

[0098] Table 1 lists the steps and / or conditions involved in affinity chromatography.

[0099]

[0100] Step (3): Hydrophobic Chromatography

[0101] a. Equilibration: The hydrophobic chromatography packing material Diamond Phenyl(HS) was equilibrated using a equilibration buffer of “30 mmol / L Tris-HCl, pH 6.5 ± 0.2”.

[0102] b. Sample loading: Load the antibody A sample prepared in step (2) above into a hydrophobic chromatography column that has been equilibrated in step a above. The column height is 15cm ± 5cm, the sample loading capacity is 10-60mg / ml, the sample retention time is 9-27min, and the unbound antibody A is washed with the equilibration buffer from step a and collected. The linear elution flow rate is 100cm / h-300cm / h.

[0103] The parameters and / or conditions involved in each step of hydrophobic chromatography are shown in Table 2.

[0104] Table 2 lists the steps and / or conditions involved in hydrophobic chromatography.

[0105]

[0106]

[0107] Step (4): Reversed phase chromatography

[0108] a. Equilibration: Equilibrate the reverse phase chromatography packing material Best Poly15RPC using an equilibration buffer solution of 11% acetonitrile aqueous solution;

[0109] b. Sample loading: Load the antibody A sample prepared in step (3) above into the reverse phase chromatography column that has been equilibrated in step a above. The column height is 15cm ± 5cm, the sample loading capacity is 5-30mg / ml, the sample retention time is 9-18min, and the unbound material is removed by rinsing with the equilibration buffer from step a. The linear elution flow rate is 100cm / h-200cm / h.

[0110] c. Elution: Elute the target protein antibody A with elution buffer “26% acetonitrile aqueous solution”.

[0111] The parameters and / or conditions involved in each step of reverse phase chromatography are shown in Table 3.

[0112] Table 3 lists the steps and / or conditions involved in reversed-phase chromatography.

[0113]

[0114] Step (5): Cation chromatography

[0115] The antibody A sample prepared in step (4) above was purified by cation chromatography. The specific steps of cation chromatography are as follows:

[0116] a. Equilibration: Equilibrate the UniGel-80SP cation exchange chromatography packing material using equilibration buffer “50mmol / L PBS, pH 6.5±0.2”;

[0117] b. Sample loading: Load antibody A from step (4) above at a loading capacity of 10-50 mg / ml, ensuring that the sample is in contact with the packing material for 9-27 min, and wash with the equilibration buffer described in step a to remove unbound material.

[0118] c. Elution: Elute antibody A after step b with elution buffer “50 mmol / L PBS and 0.3 mol / L sodium chloride, pH 6.5 ± 0.2”.

[0119] The parameters and / or conditions involved in each step of cation chromatography are shown in Table 4.

[0120] Table 4 lists the steps and / or conditions involved in cation chromatography.

[0121]

[0122] See Figures 11-13By purifying the crude antibody A using the methods described in steps (1)-(5) above, a high purity antibody A of 97% and a yield of approximately 90% can be obtained. Furthermore, the residual amount of acetonitrile is only 0.003%, and the residual amounts of impurities are all less than 1%, which is far below the pharmacopoeia requirements.

[0123] Purification method of antibody A (Comparative Example 1)

[0124] The difference between Comparative Example 1 and Example 1 is that this example does not include a cation chromatography step. While the method used in this example can achieve a purity of at least 97% for antibody A, the acetonitrile residue is 26%. This method contains a large amount of residual acetonitrile. See details... Figure 14 .

[0125] Purification method of antibody A (Comparative Example 2)

[0126] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not include a reverse phase chromatography step. When antibody A is separated and purified using the method of this example, only 88% purity antibody A can be obtained. Furthermore, the residual host protein impurity is 6.21%, which is higher than 1%, failing to meet the requirements specified in the pharmacopoeia. See details below. Figure 15 .

[0127] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for isolating and purifying a PCSK9 monoclonal antibody, characterized by, Comprise the following steps: (1) filtration, after filtering the cell culture solution of antibody A, obtaining the crude liquid of the antibody A; (2) affinity chromatography; after the crude liquid of the antibody A is treated by the affinity chromatography, the clarified liquid of the antibody A is obtained; the filler of the affinity chromatography is selected from AT ProteinA Diamond plus, the equilibrium buffer of the affinity chromatography is selected from PBS, the concentration of the equilibrium buffer is 50mmol / L, pH6.0-7.0, the elution collection buffer of the affinity chromatography is selected from Gly-HCL, the concentration of the elution collection buffer is 50mmol / L, pH2.5-4.5; (3) hydrophobic chromatography; the antibody A treated by step (2) is treated by hydrophobic chromatography; the filler of the hydrophobic chromatography is selected from Diamond Phenyl(HS), the equilibrium buffer of the hydrophobic chromatography is selected from Tris-HCL, and the concentration of the equilibrium buffer is selected from 30mmol / L, pH6.0-8.0; (4) reverse phase chromatography; the antibody A treated by step (3) is treated by reverse phase chromatography, and the filler of the reverse phase chromatography is selected from Best Poly 15RPC; the reverse phase chromatography includes an equilibrium buffer and an elution buffer, the equilibrium buffer is selected from acetonitrile, and the concentration of the acetonitrile is selected from 10%; the elution buffer is selected from acetonitrile, and the concentration of the acetonitrile is selected from 25%; (5) cation chromatography; the antibody A treated by step (4) is treated by cation chromatography to remove impurities in the antibody A, so as to obtain high-purity antibody A; the filler of the cation chromatography is selected from UniGel-80SP, the equilibrium buffer of the cation chromatography is selected from PBS, the concentration of the equilibrium buffer is selected from 50mmol / L, the elution buffer of the cation chromatography is selected from PBS and sodium chloride, the concentration of PBS in the elution buffer is 50mmol / L, the concentration of sodium chloride is 0.3mmol / L, and pH is 5.0-8.0; the antibody A is selected from antibody 18.156.

8.

2. The purification method according to claim 1, characterized in that, The affinity chromatography comprises the following steps: a. equilibrating the affinity chromatography column filled with the filler with the equilibrium buffer; b. loading: loading the crude liquid of the antibody A obtained in step (1) into the chromatography column equilibrated in step a, the loading capacity is less than or equal to 50mg / ml, the sample retention time is 9-27min, and the unbound substance is removed by flushing with the equilibrium buffer in step a; c. elution: eluting the target protein with the elution collection buffer; d. collecting the target protein treated in step c, and adjusting the pH of the target protein with the collection liquid.

3. The purification method according to claim 2, characterized in that, the collection liquid in step d is selected from at least one of Tris-HCL, PBS, MES and NaAC, and the concentration of the collection liquid is selected from 0.1-3mmol / L, and the pH is 9.0±0.

2.

4. The purification method of claim 3, wherein, the collection liquid is selected from Tris-HCL and PBS, and the concentration of the collection liquid is selected from 1mmol / L or 1.5mmol / L.

5. The purification method of claim 1, wherein, The hydrophobic chromatography consists of the following steps: a. Equilibration: equilibration of the hydrophobic chromatography packing with the equilibration buffer; b. Loading: loading of the antibody A sample prepared in step (2) above into the hydrophobic chromatography column equilibrated in step a, wherein the loading capacity is 10-60 mg / ml, the sample retention time is 9-27 min, the unbound antibody A is removed by washing with the equilibration buffer of step a, and the linear elution flow rate is 100 cm / h-300 cm / h.

6. The purification method of claim 1, wherein, The reverse phase chromatography consists of the following steps: a. Equilibration: equilibration of the reverse phase chromatography packing with the equilibration buffer; b. Loading: loading of the antibody A sample prepared in step (3) above into the reverse phase chromatography column equilibrated in step a, wherein the loading capacity is 5-30 mg / ml, the sample retention time is 9-18 min, the unbound material is removed by washing with the equilibration buffer of step a, and the linear elution flow rate is 100 cm / h-200 cm / h; c. Elution: elution of the protein of interest, antibody A, with the elution buffer.

7. The purification method of claim 1, wherein, The cationic chromatography consists of the following steps: a. Equilibration: equilibration of the cationic chromatography packing with the equilibration buffer; b. Loading: loading of the antibody A of step (4) above, wherein the loading capacity is 10-50 mg / ml, the sample is contacted with the packing for 9-27 min, and the unbound material is removed by washing with the equilibration buffer of step a; c. Elution: elution of the antibody A treated in step b with the elution buffer.

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