Cleaning method capable of obviously prolonging service life of affinity chromatography agarose base frame filler with protein A tag

Abstract

The invention discloses a cleaning method capable of obviously prolonging the service life of affinity chromatography agarose base frame filler with a protein A tag, and belongs to the technical field of biology. The decyl glucoside is added into the cleaning solution of the affinity chromatography column, so that pollutants can be effectively removed, and the mass transfer efficiency after the filler is circularly used for multiple times is maintained. Compared with cleaning without adding decyl glucoside, the method has the advantages that the target protein yield is increased from 60.9% to 87% or above after 100 cycles, and the service life of the filler is obviously prolonged.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cleaning method capable of significantly extending the service life of an affinity chromatography agarose frame filler with a protein A tag. Background Art

[0002] As an important downstream purification chromatography medium in the field of crude drug technology, affinity chromatography fillers with protein A tags can efficiently capture biomacromolecules with Fc fragments through specific and selective affinity. Usually, the purity of the target protein in the collected liquid (SEC-HPLC method) can reach more than 90%, which greatly reduces the difficulty of impurity removal in the subsequent purification steps and further ensures a higher product recovery rate downstream. Therefore, it is often used in the capture / rough purification steps of macromolecules with Fc fragments such as antibodies and recombinant proteins.

[0003] Although protein A tag affinity chromatography fillers have excellent purification effects and high sample loading capacity, they have always been an important downstream cost part in the biotechnology field due to their high manufacturing costs. In addition, in recent years, with the continuous improvement of upstream cell culture technology, the expression level of target proteins has increased significantly. At the same time, the content of pollutants in the fillers in the upstream feed liquid has also shown an increasing trend, resulting in increasing challenges in reducing the service life of affinity chromatography fillers and corresponding increases in downstream costs. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a cleaning method that can significantly extend the service life of agarose-based frame filler for affinity chromatography with a protein A tag. That is, in the step of flushing the filler during the chromatography process, a special buffer is added for flushing to specifically remove impurities attached to the filler that are difficult to remove, thereby significantly extending the service life of the protein A-tagged affinity chromatography filler to meet the demand for reducing production costs.

[0005] The present invention provides a cleaning method that can significantly extend the service life of agarose-based filler for affinity chromatography with a protein A tag, and the specific steps include:

[0006] (1) Clean the chromatography column in situ with 0.5 mol / L NaOH solution;

[0007] (2) Clean the chromatography column in situ with 0.1 mol / L NaOH solution;

[0008] (3) The affinity chromatography column is then equilibrated with Tris-HAc-NaCl buffer until the conductivity, pH, and UV absorbance of the column are stable;

[0009] (4) loading the clarified filtered collected liquid onto an affinity chromatography column, and adsorbing and binding the target protein through the affinity between the filler ligand and the Fc fragment of the target protein;

[0010] (5) After the sample is loaded, the affinity chromatography column is equilibrated with Tris-HAc-NaCl buffer to remove some impurity proteins that are not bound or weakly bound;

[0011] (6) Washing the column with NaAc-HAc-NaCl high salt buffer to wash away strongly bound impurities and DNA residues;

[0012] (7) flushing the chromatography column with NaAc-HAc buffer so that the target protein contacts the buffer system close to the elution buffer;

[0013] (8) Use NaAc-HAc low pH buffer for elution, start collecting when the 280nm UV absorbance value rises to 100mAu / 2mm, and end collecting when it decreases to 100mAu / 2mm, so that high-purity target protein can be eluted;

[0014] (9) Regenerating the chromatography column with 1 mol / L HAc buffer to clean impurities;

[0015] (10) balancing the affinity chromatography column with Tris-HAc-NaCl buffer until the conductivity, pH, and UV absorbance baseline of the chromatography column are stable;

[0016] (11) washing the chromatography column with Tris-HAc-NaCl-decyl glucoside buffer;

[0017] (12) balancing the affinity chromatography column with Tris-HAc-NaCl buffer until the conductivity, pH, and UV absorbance baseline of the chromatography column are stable;

[0018] (13) Clean the chromatography column in situ with 0.1 mol / L NaOH solution;

[0019] (14) balancing the affinity chromatography column with Tris-HAc-NaCl buffer until the conductivity, pH, and UV absorbance baseline of the chromatography column are stable;

[0020] (15) Preserve the affinity chromatography column with 20% ethanol;

[0021] The affinity chromatography columns involved in the above steps (1) to (15) all represent chromatography columns filled with affinity chromatography agarose-based frame fillers.

[0022] Furthermore, in step (1), the in-situ cleaning of the chromatography column with 0.4-0.6 mol / L NaOH solution is performed once for 8-12 cycles.

[0023] Preferably, in step (1), the chromatography column is cleaned in situ with a 0.5 mol / L NaOH solution, and the cleaning is performed once every 10 cycles.

[0024] Furthermore, in step (1), the volume ratio of the chromatography column filler to 0.5 mol / L NaOH is (4-6):14.28.

[0025] Preferably, in step (1), the volume ratio of the chromatography column filler to 0.5 mol / L NaOH is 4.76:14.28.

[0026] Furthermore, in step (1), the retention time of the 0.5 mol / L NaOH solution is 3 to 7 minutes.

[0027] Preferably, the retention time of the 0.5 mol / L NaOH solution in step (1) is 5 min.

[0028] Furthermore, in step (3), the Tris concentration in the Tris-HAc-NaCl buffer is 40-60 mmol / L, the NaCl concentration is 100-200 mmol / L, and the pH is 7-8.

[0029] Preferably, in step (3), the Tris concentration of the Tris-HAc-NaCl buffer is 50 mmol / L, the NaCl concentration is 150 mmol / L, and the pH is 7.4.

[0030] Furthermore, in step (3), the volume ratio of the chromatography column filler and the Tris-HAc-NaCl buffer is (4-6):14.28.

[0031] Preferably, in step (3), the volume ratio of the chromatography column filler to the Tris-HAc-NaCl buffer is 4.76:14.28.

[0032] Furthermore, the retention time of the Tris-HAc-NaCl buffer in step (3) is 3 to 7 minutes.

[0033] Preferably, the retention time of the Tris-HAc-NaCl buffer in step (3) is 5 min.

[0034] Furthermore, the clarified filtered collected liquid buffer in step (4) is a sample obtained by centrifuging or clarifying the cell suspension after upstream CHO cell culture.

[0035] Furthermore, in step (4), the retention time of the clarified filtered collected liquid loaded onto the chromatography column is 3 to 7 minutes.

[0036] Preferably, in step (4), the retention time of the clarified filtered collected liquid loaded onto the chromatography column is 5 min.

[0037] Furthermore, step (5) is the same as step (3).

[0038] Furthermore, in step (6), the NaAc-HAc concentration in the NaAc-HAc-NaCl buffer is 40-60 mmol / L, the NaCl concentration is 0.9-1.1 mol / L, and the pH is 4.8-5.2.

[0039] Preferably, in step (6), the NaAc-HAc concentration of the NaAc-HAc buffer is 50 mmol / L, the NaCl concentration is 1 mol / L, and the pH is 5.0.

[0040] Furthermore, in step (6), the volume ratio of the chromatography column to the NaAc-HAc-NaCl buffer is (4-6):14.28.

[0041] Preferably, in step (6), the volume ratio of the chromatography column to the NaAc-HAc-NaCl buffer is 4.76:14.28.

[0042] Furthermore, the retention time of the NaAc-HAc-NaCl buffer in step (6) is 3 to 7 minutes.

[0043] Preferably, the retention time of the NaAc-HAc-NaCl buffer in step (6) is 5 min.

[0044] Furthermore, in step (7), the concentration of NaAc-HAc in the NaAc-HAc buffer is 40 to 60 mmol / L, and the pH is 4.8 to 5.2.

[0045] Preferably, in step (7), the NaAc-HAc buffer has a NaAc-HAc concentration of 50 mmol / L and a pH of 5.0.

[0046] Furthermore, in step (7), the volume ratio of the chromatography column to the NaAc-HAc buffer is (4-6):14.28.

[0047] Preferably, in step (7), the volume ratio of the chromatography column to the NaAc-HAc buffer is 4.76:14.28.

[0048] Furthermore, the retention time of the NaAc-HAc buffer in step (7) is 3 to 7 minutes.

[0049] Preferably, the retention time of the NaAc-HAc buffer in step (7) is 5 min.

[0050] Furthermore, in step (8), the concentration of NaAc-HAc in the NaAc-HAc buffer is 40 to 60 mmol / L, and the pH is 3.6 to 3.9.

[0051] Preferably, in step (8), the NaAc-HAc concentration in the NaAc-HAc buffer is 50 mmol / L and the pH is 3.75.

[0052] Furthermore, in the NaAc-HAc-NaCl-decyl glucoside buffer in step (11), the concentration of NaAc-HAc is 40-60 mmol / L, the concentration of NaCl is 100-200 mmol / L, the concentration of decyl glucoside is 0.5-1.5%, and the pH is 7-8.

[0053] Preferably, in the NaAc-HAc-NaCl-decyl glucoside buffer in step (11), the concentration of NaAc-HAc is 50 mmol / L, the concentration of NaCl is 150 mmol / L, the concentration of decyl glucoside is 1.0%, and the pH is 7.4.

[0054] Furthermore, in step (11), the volume ratio of the chromatography column to the NaAc-HAc-NaCl-decyl glucoside buffer is (4-6):14.28.

[0055] Preferably, in step (11), the volume ratio of the chromatography column to NaAc-HAc-NaCl-decyl glucoside is 4.76:14.28.

[0056] Furthermore, in step (11), the retention time of NaAc-HAc-NaCl-decyl glucoside is 3 to 7 minutes.

[0057] Preferably, the retention time of NaAc-HAc-NaCl-decyl glucoside in step (11) is 5 min.

[0058] Furthermore, in steps (10), (12) and (14), the volume ratio of the chromatography column to the NaAc-HAc-NaCl buffer solution is 4.76:9.52, and the rest is the same as step (3).

[0059] Furthermore, step (13) is the same as step (2).

[0060] The present invention also provides application of the method in prolonging the service life of an affinity chromatography agarose frame filler with a protein A tag.

[0061] Beneficial effects:

[0062] In the capture step of affinity chromatography with protein A tag, the traditional NaOH buffer system is used for in-situ cleaning, which cannot effectively clean the dirt adsorbed deep in the filler, resulting in a significant weakening of the filler affinity as the number of times the affinity filler is used increases (as can be seen from the dynamic binding capacity determination curve of the filler, see Figure 3), resulting in a decrease in the packing loading and a significant decrease in the recovery rate of the target protein; by adding the decyl glucoside buffer to the flushing step, the dirt deep in the packing can be effectively flushed, which can significantly extend the service life of the packing (it can remove pollutants and maintain the mass transfer efficiency of the packing after multiple cycles of use, see Figure 3 ), for the control group (without decyl glucoside buffer washing) using the same 100 cycles, the yield of the target protein increased from 60.9% to more than 87%. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Overlays of 1-cycle and 100-cycle chromatograms were used for different experimental groups;

[0064] Figure 2 The yield curves of different experimental groups;

[0065] Figure 3 This is the comparison curve of dynamic load determination of different cycles between the control group and experimental group 1;

[0066] Figure 4 Comparison curves of dynamic loading capacity determination in different cycles of experimental group 1 and experimental group 4;

[0067] Among them, the control group represents AT Protein ADiamond filler, and step 11 is Tris-HAc-NaCl buffer (Comparative Example 1); experimental group 1 represents AT Protein ADiamond filler, and step 11 is Tris-HAc-NaCl-decyl glucoside buffer (Example 1); experimental group 2 represents MaXtar ARPA filler, and step 11 is Tris-HAc-NaCl-decyl glucoside buffer (Example 2); experimental group 3 represents NMab Pro filler, and step 11 is Tris-HAc-NaCl-decyl glucoside buffer (Example 3); experimental group 4 represents AT Protein A Diamond filler, and step 11 is Tris-HAc-NaCl-TritonX-100 buffer (Example 4). DETAILED DESCRIPTION

[0068] Source of raw materials

[0069] Clarify and filter the collected liquid (the liquid containing the target protein after the upstream CHO cell culture is harvested and passed through the clarifying filtration membrane).

[0070] AT Protein ADiamond filler was provided by Boglon (Zhejiang) Biotechnology Co., Ltd. (Cat. No.: AA0276); MaXtar ARPA filler was provided by Bailinke (Lanzhou) New Materials Co., Ltd. (Cat. No.: 1024-1824); NMab Pro filler was provided by Suzhou Nanotech Co., Ltd. (Cat. No.: 17013-070100-1005). Triton X-100 was provided by Sigma-Aldrich (Wuxi) Biochemical Technology Co., Ltd. (Cat. No.: T9284-1L); Decyl glucoside was provided by Guangdong Xiaoda Chemical Co., Ltd. (Cat. No.: 141464-42-8).

[0071] The concentration percentages in the following examples, unless otherwise specified, refer to mass percentages. For example, 1.0% decyl glucoside means taking 10 g of decyl glucoside and weighing it to 1000 g with ultrapure water.

[0072] Example 1

[0073] (1) Use 14.28 mL of 0.5 mol / L NaOH to clean a 4.76 mL AT Protein ADiamond column in place (once for every 10 cycles of using the filler), with a retention time of 5 min for each cleaning;

[0074] (2) flushing the ATProteinADiamond column with 14.28 mL 0.1 mol / L NaOH for in-situ cleaning with a retention time of 5 min;

[0075] (3) The AT Protein ADiamond column was then equilibrated with 14.28 mL of a buffer solution containing 50 mmol / L Tris-HAc and 150 mmol / L NaCl at pH 7.4, with a retention time of 5 min, until the conductivity, pH, and UV absorbance baselines of the column were stable;

[0076] (4) The clarified filtered collected liquid is loaded onto the AT Protein ADiamond chromatography column, and the target protein is adsorbed and bound by the affinity between the filler ligand and the target protein, with a retention time of 5 minutes;

[0077] (5) After loading, the ATProteinADiamond column was equilibrated with 14.28 mL of a buffer solution containing 50 mmol / L Tris-HAc and 150 mmol / L NaCl, pH 7.4, to remove some impurity proteins that were not bound or weakly bound;

[0078] (6) The ATProteinADiamond column was washed with 14.28 mL of a buffer solution containing 50 mmol / L NaAc-HAc and 1 mol / L NaCl, pH 5.0, to wash away strongly bound impurities and DNA residues, with a retention time of 5 min;

[0079] (7) Rinse the ATProteinADiamond column with 14.28 mL of 50 mmol / L NaAc-HAc, pH 5.0 buffer so that the target protein contacts the buffer system close to the elution buffer, with a retention time of 5 min;

[0080] (8) Elution was performed with 23.66 mL of 50 mmol / L NaAc-HAc, pH 3.75 buffer. Collection began when the UV absorbance value rose to 100 mAu / 2 mm and ended when it dropped to 100 mAu / 2 mm. Highly pure target protein could be eluted.

[0081] (9) Regenerating the chromatography column with 14.28 mL of 1 mol / L HAc buffer to clean impurities;

[0082] (10) The ATProtein ADiamond column was equilibrated with 9.52 mL of a buffer solution containing 50 mmol / L Tris-HAc and 150 mmol / L NaCl, pH 7.4, with a retention time of 5 min until the conductivity, pH, and UV absorbance baselines of the column were stable;

[0083] (11) regenerating the ATProteinADiamond column with 14.28 mL of a buffer solution containing 50 mmol / L Tris-HAc, 150 mmol / L NaCl, and 1.0% decyl glucoside, pH 7.4, with a retention time of 5 min;

[0084] (12) The ATProtein ADiamond column was equilibrated with 9.52 mL of a buffer solution containing 50 mmol / L Tris-HAc and 150 mmol / L NaCl, pH 7.4, with a retention time of 5 min until the conductivity, pH, and UV absorbance baselines of the column were stable;

[0085] (13) The ATProteinADiamond column was rinsed with 14.28 mL of 0.1 mol / L NaOH for in-situ cleaning with a retention time of 5 min;

[0086] (14) The ATProteinA Diamond column was equilibrated with 9.52 mL of 50 mmol / L Tris-HAc, 150 mmol / L NaCl, pH 7.4 buffer with a retention time of 5 min until the conductivity, pH, and UV absorbance of the column were stable;

[0087] (15) Use 14.28 mL of 20% ethanol to preserve the AT Protein ADiamond chromatography column.

[0088] The results showed that after 100 cycles, the chromatography yield was 87.4% ( Figure 2 ).

[0089] Embodiments 2 to 4

[0090] The experimental steps are the same as those in Example 1, except that the chromatographic column in Example 2 is filled with MaXtarARPA, and the results show that after 100 cycles, the yield is 87.6%; the chromatographic column in Example 3 is filled with NMab Pro, and the results show that after 100 cycles, the yield is 88.9%; Step 11 in Example 4 is to regenerate the AT Protein ADiamond chromatographic column with 14.28 mL of a buffer solution containing 50 mmol / L Tris-HAc, 150 mmol / L NaCl and 1.0% TritonX-100, pH 7.4, with a retention time of 5 min. The results show that after 100 cycles, the yield is 72.5% ( Figure 2 ).

[0091] Comparative Example 1:

[0092] Specific implementation method is the same as Example 1, except that step (11) is replaced by equilibrating the chromatography column with 14.28 mL 50 mmol / LTris-HAc, 150 mmol / LNaCl, pH 7.4 buffer.

[0093] The results showed that: under this chromatography mode, after executing 100 cycles, the chromatography yield was only 60.9%. Figure 2 shown.

[0094] Combination Figure 1 ,Compared with experimental group 1, the control group filler used 100 cycles, the protein flow-through was serious near the end of the loading stage, and the UV absorption peak was larger during the rinsing stage;

[0095] Combination Figure 3 As the number of times the filler was used increased, when the sample loading was 20 g / L to 40 g / L, the flowthrough of the target protein in the control group was significantly higher than that in the experimental group 1.

[0096] In summary, after 100 cycles of chromatography, the yield of experimental group 1 (87.4%) was significantly higher than that of the control group (60.9%), which proved that flushing the chromatography column with decyl glucoside can significantly improve the service life of the filler.

[0097] Combination Figure 1 ,Compared to experimental group 1, the filler in experimental group 4 used 100 cycles, and the UV absorption peak was larger during the rinsing stage.

[0098] Combination Figure 4 As the number of times the filler is used increases, when the sample loading capacity is 20g / L~40g / L, the protein breakthrough of experimental group 4 is higher than that of experimental group 1.

[0099] In summary, after 100 cycles of chromatography, the yield of experimental group 1 (87.4%) was significantly higher than that of experimental group 4 (72.5%), proving that the use of decyl glucoside to wash the chromatography column is better than TritonX-100 in improving the service life of the filler.

[0100] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for cleaning agarose-based filler with protein A tag affinity chromatography, characterized in that: Cleanse with Decyl Glucoside.

2. The method according to claim 1, characterized in that: The method comprises the following steps: (1) Clean the chromatography column in situ with 0.4-0.6 mol / L NaOH solution; (2) Clean the column in situ with 0.08-0.12 mol / L NaOH solution; (3) equilibrate the affinity chromatography column with Tris-HAc-NaCl buffer; (4) loading the sample onto an affinity chromatography column; (5) equilibrate the affinity chromatography column with Tris-HAc-NaCl buffer after loading the sample; (6) washing the chromatography column with NaAc-HAc-NaCl high salt buffer; (7) washing the chromatography column with NaAc-HAc buffer; (8) elution with NaAc-HAc low pH buffer; (9) Regenerating the chromatography column with 0.5-1.5 mol / L HAc buffer; (10) equilibrate the affinity chromatography column with Tris-HAc-NaCl buffer; (11) washing the chromatography column with Tris-HAc-NaCl-decyl glucoside buffer; (12) equilibrate the affinity chromatography column with Tris-HAc-NaCl buffer; (13) flushing the affinity chromatography column with 0.08-0.12 mol / L NaOH solution for in-situ cleaning; (14) Equilibrate the affinity chromatography column with Tris-HAc-NaCl buffer.

3. The method according to claim 2, characterized in that: In the buffer solution of step (11), the concentration of Tris is 40-60 mmol / L, the concentration of NaCl is 100-200 mmol / L, and the mass percentage of decyl glucoside is 0.5-1.5%.

4. The method according to claim 2, characterized in that: In step (11), the retention time of the buffer solution in the chromatography column is 3 to 7 minutes.

5. The method according to claim 2, characterized in that: In step (11), the pH of the buffer solution is 7.2 to 7.

6.

6. The method according to claim 2, characterized in that: The high salt buffer in step (6) contains 40-60 mmol / L HAc and 0.9-1.1 mol / L NaCl.

7. The method according to claim 2, characterized in that: The pH of the buffer solution in step (8) is 3.6 to 3.

9.

8. The method according to claim 2, characterized in that: The buffer solution in step (10) and step (14) contains 40-60 mmol / L Tris and 100-200 mmol / L NaCl.

9. Use of the method according to any one of claims 1 to 8 in extending the service life of agarose-based support filler for affinity chromatography with a protein A tag.

10. Use of the method according to any one of claims 1 to 8 in improving the purification yield of affinity chromatography agarose-based support material with a protein A tag.

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