High-efficiency cell supernatant Protein A purification method

Abstract

The invention discloses a high-efficiency cell supernatant Protein A purification method, and belongs to the technical field of biology. A porous filter plate (1) is used for replacing a pre-assembled column to serve as a purification filler supporting medium, protein A filler is loaded into a hole site, gaskets of 10 micrometers are placed above and below the filler, the gaskets do not adsorb protein, the filler can be prevented from floating when a sample is added, the uniformity of the thickness of the filler is guaranteed, sufficient combination of protein and the filler is guaranteed, and the protein recovery rate is increased. And filtering the cell supernatant by using a 0.45 mu m filter membrane, adding the filtered cell supernatant into a porous filter plate (1) containing protein A filler, and carrying out impurity cleaning and elution to collect the target protein. Compared with a preassembled column purification mode, the method has the advantages that single-time multi-flux filtration can be realized by using the vacuum pump, the purification efficiency is high, and the consumed time is short; compared with a purification mode of a protein purification instrument, the method is simple in material consumption, low in cost, short in time consumption and suitable for high-throughput sample treatment.

Description

Technical Field

[0001] The invention relates to a high-efficiency method for purifying cell supernatant Protein A, and belongs to the field of biotechnology. Background Art

[0002] Protein A is a membrane protein of Staphylococcus aureus, which has the ability to specifically bind to antibodies. Protein A affinity chromatography column has become a widely used affinity column for purifying antibodies. Protein A contains 5 domains that can specifically bind to the Fc segment of antibody IgG molecules, which enables it to specifically bind to antibody molecules in the sample while allowing other impurities to flow through. It has extremely high selectivity and is usually used in the protein capture step.

[0003] The protein A purification process includes equilibration, loading, impurity elution, elution, strong washing, and regeneration. During the purification process, the protein yield and purification efficiency requirements need to be considered.

[0004] There are two common ways to purify cell supernatant. One is to use a protein A pre-packed column with a peristaltic pump or syringe to purify cell supernatant protein; the other is to use a commercial protein A column with a protein purifier for purification. The former is low-cost and easy to purchase and install by yourself, suitable for the purification of small samples, and has high flexibility; the latter has expensive instrument consumables and high purification costs, and is suitable for the purification of large amounts of cell supernatant samples.

[0005] However, the above technology has the following problems: in the development process of CHO cell culture medium, DOE experiments and multi-cell verification shake flask experiments are involved, and the number of shake flasks is as many as 200. A single pre-loaded column or protein purifier cannot quickly complete the purification of all cell supernatant samples, and the efficiency is low. Although increasing the number of pre-loaded columns or protein purifiers can increase the purification flux, it also leads to an increase in the number of operators, which increases the manpower and material costs, and cannot meet the dual demands for protein purification efficiency and cost. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a simple and high-throughput method for purifying cell supernatant Protein A, which aims to solve the technical problems that the existing single pre-loaded column or protein purifier cannot quickly complete the purification of all cell supernatant samples, has high manpower and material costs, and has low efficiency.

[0007] The first technical solution provided by the present invention is a highly efficient method for purifying protein A from cell supernatant, comprising the following steps:

[0008] S1, making a Protein A purification filter plate: put a 10μm pore size lower gasket at the bottom of the porous filter plate. This gasket has no protein adsorption and can prevent protein A filler leakage. Load protein A filler into each well, and put a 10μm pore size upper gasket on top of the filler. This gasket has no protein adsorption and can prevent the filler from floating when the sample is added, ensuring the uniformity of the filler thickness, ensuring that the protein and the filler are fully combined, and improving the protein recovery rate;

[0009] S2, cell supernatant purification: place the sample receiving plate in the base, and then stack the Protein A purification filter plate in S1 on the base to purify the cell supernatant, including equilibration, loading, impurity elution, target protein elution, strong washing and regeneration. In order to increase the speed of buffer addition, PB buffer (pH7.0), acetic acid solution (50mM, for reference, adjustable), citric acid solution (100mM, for reference, adjustable) and sodium hydroxide solution (100mM, for reference, adjustable) can be added to the porous filter plate using a discharge gun or a wash bottle. After the solution is added, use a gas supply device to filter to increase the purification rate.

[0010] In certain embodiments, each cavity of the porous filter plate has a porous filter plate at the bottom and is provided with a liquid outlet.

[0011] In certain embodiments, the sample receiving plate is provided with a plurality of liquid receiving cavities, and the plurality of liquid outlets are aligned with the plurality of liquid receiving cavities.

[0012] In some embodiments, a placement cavity is provided in the base, and an air hole connected to the air supply device is opened on the side.

[0013] In certain embodiments, the purification process is as follows:

[0014] Balance: Add PB buffer into each cavity of the porous filter plate and filter;

[0015] Loading: Add cell supernatant into each cavity of the porous filter plate and filter;

[0016] Elution of impurities: Add PB buffer into each cavity of the porous filter plate and perform suction filtration;

[0017] Elution: Add acetic acid solution into each cavity of the porous filter plate, filter and collect the filtrate, i.e. the target protein;

[0018] Strong washing: add citric acid solution into each cavity of the porous filter plate and filter;

[0019] Regeneration: Add sodium hydroxide solution into each cavity of the porous filter plate and filter it.

[0020] Furthermore, in the equilibration step, the pH of the PB buffer was 7.0, and the amount added was 4 to 6 mL (greater than 3 times the volume of the filler).

[0021] Furthermore, in the loading step, the cell supernatant is prepared by centrifuging the cells after cell disruption, collecting the supernatant and filtering it with a 0.45 μm filter membrane.

[0022] Furthermore, in the impurity elution step, the pH of the PB buffer was 7.0, and the amount added was 4 to 6 mL (greater than 3 times the volume of the filler).

[0023] Furthermore, in the elution step, the concentration of the acetic acid solution is 50 mM, and the amount added is 4 to 6 mL (greater than 3 times the volume of the filler).

[0024] Furthermore, in the strong washing step, the concentration of citric acid was 100 mM.

[0025] Furthermore, in the regeneration step, the concentration of sodium hydroxide was 100 mM.

[0026] Furthermore, in the loading and elution steps, after adding the solution or sample, wait for 5 minutes, and then use a gas supply device to generate a gas pressure difference for suction filtration.

[0027] In certain embodiments, the gas supply device is a vacuum pump.

[0028] The second technical solution provided by the present invention is the application of the method described in the first technical solution in the purification of cell supernatant with high protein content.

[0029] Beneficial Effects

[0030] The present invention uses a porous filter plate instead of a pre-loaded column as a support medium for purification filler, and a 10 μm gasket is placed at the bottom of the porous filter plate. The gasket has no protein adsorption and can prevent leakage of the protein A filler. The protein A filler is loaded into the hole position, and a 10 μm gasket is placed above the filler. The gasket has no protein adsorption, which can prevent the filler from floating when the sample is added, ensure the uniformity of the filler thickness, ensure that the protein and the filler are fully combined, and improve the protein recovery rate. During purification, gas is used to apply pressure to the added liquid for suction filtration (the pressure can be positive pressure or negative pressure, and the gas supply device can be a vacuum pump, a large volume syringe, etc.), thereby increasing the purification speed. After purification is completed, the filter plate can be split according to demand to recover the filler and gasket.

[0031] The present invention is suitable for purifying proteins containing Fc regions, and any sample containing proteins can be used in the present invention. The cell supernatant is filtered using a 0.45 μm filter membrane, and the filtered sample can be added to a porous filter plate containing protein A filler, and the target protein can be collected after impurity cleaning and elution steps. Compared with the pre-loaded column purification method, the present invention uses gas instead of a peristaltic pump or a syringe to achieve single-time multi-flux filtration, with high purification efficiency and short time; compared with the protein purifier purification method, the present invention has simple consumables, low cost, high purification efficiency, short time, and is suitable for the processing of high-throughput samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The figure is a flow chart of the method for purifying protein A in cell supernatant of the present invention.

[0033] Figure 2 Schematic diagram of a 24-well filter plate.

[0034] Figure 3 The sample receiving plate.

[0035] Figure 4 The base has air holes.

[0036] Figure 5 Schematic diagram of the use of 24-well filter plate, sample receiving plate and base. DETAILED DESCRIPTION

[0037] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0038] The raw materials used in the embodiment:

[0039] 1. The raw materials used in the present invention include Protein A filler purchased from AT Protein ADiamond Plus of Boglon. The cell supernatant expressed by CHO-K1 cell line, the cell line is from ATCC cell bank, item number CBP60296. The cell supernatant in implementation case 1 is the 10-day harvest liquid of Batch culture process, and the cell supernatant in implementation case 2 is the 14-day harvest liquid of Fed-Batch culture process.

[0040] Example 1 High-efficiency cell supernatant purification

[0041] like Figure 1 As shown, 24 filtered samples are purified simultaneously using a multi-well filter plate (a 24-well filter plate is used in this case, and a 12-well or 48-well filter plate can also be selected according to needs), including the following steps:

[0042] S1, making a Protein A purification filter plate: placing a 10 μm pore size lower gasket at the bottom of the porous filter plate 1. The gasket has no protein adsorption and can prevent leakage of the protein A filler. 1 mL of protein A filler is loaded into each well. A 10 μm pore size upper gasket is placed above the filler. The gasket has no protein adsorption and can prevent the filler from floating when the sample is added, thereby ensuring uniform thickness of the filler, ensuring full combination of the protein and the filler, and improving the protein recovery rate.

[0043] S2, cell supernatant purification: Figure 5 As shown, the sample receiving plate 2 is placed in the base 3, and then the Protein A purification filter plate in S1 is stacked on the base 3 and located above the sample receiving plate 2. The purification of the cell supernatant includes 6 steps, namely, equilibration, loading, impurity elution, target protein elution, strong washing, and regeneration. In order to increase the speed of adding buffer, PB buffer (pH 7.0), acetic acid solution (50mM), citric acid solution (100mM,) and sodium hydroxide solution (100mM) can be added to the porous filter plate using a discharge gun or a wash bottle. After the solution is added, a vacuum pump can be used for filtration to improve purification efficiency. The purification process is as follows:

[0044] Balance: Add 5 mL of PB buffer (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0045] Loading: Add 5 mL of cell supernatant to each well of the multi-well filter plate (repeated loading is possible) and filter using a vacuum pump;

[0046] Elution of impurities: Add 5 mL of PB buffer (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0047] Elution: Add 5 mL of acetic acid solution (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate, use a vacuum pump to filter and collect the filtrate, which is the target protein;

[0048] Strong wash: add 5 mL of citric acid solution (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0049] Regeneration: Add 5 mL of sodium hydroxide solution (buffer volume ≥ 3 times the filler volume) to each hole of the porous filter plate and filter using a vacuum pump.

[0050] like Figure 2 As shown, each cavity 11 of the porous filter plate 1 has a filter plate 12 at the bottom and a liquid outlet 13 .

[0051] like Figures 3-4As shown, the sample receiving plate 2 is provided with a plurality of liquid receiving cavities 21, and the plurality of liquid outlets 13 are aligned with the plurality of liquid receiving cavities 21. The base 3 is provided with a placement cavity 31, and a gas hole 32 connected to a gas supply device (vacuum pump) is opened on the side.

[0052] It took a total of 59 minutes to purify 24 samples using a 24-well filter plate. The time for each step is shown in the following table. Using a pre-packed column, it took about 15 minutes to purify each sample, and it took about 360 minutes for 24 samples. Using a protein purifier, it took about 25 minutes to purify each sample, and it took about 600 minutes for 24 samples. In the case of a single operator, the use of a porous filter plate to purify protein has a higher efficiency.

[0053] Table 1 Statistics of time required to purify 24 samples using porous filter plates

[0054]

[0055]

[0056] After purification, the antibody yield of the samples was determined by Elisa to calculate the recovery rate. There was no significant difference in the recovery rate between the porous filter plate purification method and the pre-packed column purification method. The charge heterogeneity test results showed that there was no significant difference between the two purification methods.

[0057] Table 2 Purification yield

[0058] Purification method Amount of protein loaded (mg) Amount of recovered protein (mg) Yield Prepacked columns 28.5 23.4 89％ Porous filter plate 28.5 26.2 92％

[0059] Table 3 Charge heterogeneity test results of purified samples

[0060]

[0061] Example 2 Purification of high concentration cell supernatant

[0062] 1mL of filler can usually fix 30mg of protein. Taking 5mL of cell supernatant as an example, when the cell supernatant protein concentration is greater than 6g / L, the filler volume can be increased to fix more protein. This case shows the purification method for a high-concentration protein of 9g / L. The steps are as follows:

[0063] S1, making a Protein A purification filter plate: put a 10μm pore size lower gasket at the bottom of the porous filter plate. This gasket has no protein adsorption and can prevent protein A filler leakage. Load 2mL of protein A filler into each well, and put a 10μm pore size upper gasket on top of the filler. This gasket has no protein adsorption and can prevent the filler from floating when the sample is added, ensuring the uniformity of the filler thickness, ensuring that the protein and the filler are fully combined, and improving the protein recovery rate;

[0064] S2, set up the sample receiving plate on the base, and then stack the Protein A purification filter plate in S1 on the sample receiving plate to purify the cell supernatant: cell supernatant purification includes 6 steps: equilibration, loading, impurity elution, target protein elution, strong washing, and regeneration. In order to increase the speed of buffer addition, PB buffer (pH7.0), acetic acid solution (50mM, for reference, adjustable), citric acid solution (100mM, for reference, adjustable) and sodium hydroxide solution (100mM, for reference, adjustable) can be added to the porous filter plate using a discharge gun or a wash bottle. After the solution is added, a vacuum pump can be used for filtration to provide purification efficiency. The purification process is as follows:

[0065] Balance: Add 5 mL of PB buffer (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0066] Loading: Add 5 mL of cell supernatant to each well of the multi-well filter plate (repeated loading is possible) and filter using a vacuum pump;

[0067] Elution of impurities: Add 5 mL of PB buffer (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0068] Elution: Add 5 mL of acetic acid solution (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate, use a vacuum pump to filter and collect the filtrate, which is the target protein;

[0069] Strong wash: add 5 mL of citric acid solution (buffer volume ≥ 3 times the filler volume) to each well of the porous filter plate and filter using a vacuum pump;

[0070] Regeneration: Add 5 mL of sodium hydroxide solution (buffer volume ≥ 3 times the filler volume) to each hole of the porous filter plate and filter using a vacuum pump.

[0071] It took a total of 59 minutes to purify 24 samples using a 24-well filter plate. The time for each step is shown in the following table. Using a pre-packed column, it took about 15 minutes to purify each sample, and it took about 360 minutes for 24 samples. Using a protein purifier, it took about 25 minutes to purify each sample, and it took about 600 minutes for 24 samples. In the case of a single person operating, the use of a porous filter plate to purify protein has a higher efficiency.

[0072] 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 highly efficient method for purifying protein A from cell supernatant, characterized in that: The steps include: S1, making a Protein A purification filter plate: placing a 10 μm pore size lower gasket at the bottom of the porous filter plate (1), the gasket has no protein adsorption and can prevent protein A filler leakage, loading protein A filler into each cavity (11), and placing a 10 μm pore size upper gasket on top of the filler, the gasket has no protein adsorption and can prevent the filler from floating when the sample is added, ensuring the uniformity of the filler thickness, ensuring that the protein and the filler are fully combined, and improving the protein recovery rate; S2, cell supernatant purification: place the sample receiving plate (2) in the base (3), and then stack the Protein A purification filter plate in S1 on the base (3) and place it above the sample receiving plate (2), and purify the cell supernatant, including equilibration, sample loading, impurity elution, target protein elution, strong washing and regeneration.

2. The purification method according to claim 1, characterized in that The bottom of each cavity (11) of the porous filter plate (1) is provided with a filter plate (12) and a liquid outlet (13), and the lower gasket is placed on the filter plate (12).

3. The purification method according to claim 1, characterized in that The sample receiving plate (2) is provided with a plurality of liquid receiving cavities (21), and the plurality of liquid outlets (13) are aligned with the plurality of liquid receiving cavities (21).

4. The purification method according to claim 1, characterized in that The base (3) is provided with a placement cavity (31) in the interior, and an air hole (32) connected to the air supply device is opened on the side.

5. The purification method according to claim 1, characterized in that The purification process is as follows: Balance: adding PB buffer to each cavity (11) of the porous filter plate (1) and filtering; Loading: adding cell supernatant into each cavity (11) of the porous filter plate (1) and filtering; Elution of impurities: adding PB buffer to each cavity (11) of the porous filter plate (1) and performing suction filtration; Elution: adding acetic acid solution into each cavity (11) of the porous filter plate (1), filtering and collecting the filtrate, i.e., the target protein; Strong washing: adding citric acid solution into each cavity (11) of the porous filter plate (1) and performing suction filtration; Regeneration: Add sodium hydroxide solution into each cavity (11) of the porous filter plate (1) and perform suction filtration.

6. The purification method according to claim 5, characterized in that During the equilibration step, the pH of the PB buffer was 7.0, and the amount added was at least 3 times the volume of the filler; In the impurity elution step, the pH of the PB buffer was 7.0, and the amount added was at least 3 times the volume of the filler; In the elution step, the concentration of acetic acid solution was 50 mM, and the amount added was at least 3 times the volume of the filler; In the strong wash step, the concentration of citric acid was 100 mM; During the regeneration step, the concentration of sodium hydroxide was 100 mM.

7. The method according to claim 5, characterized in that Preparation of cell supernatant: After cell disruption, centrifugation was performed, and the supernatant was collected and filtered through a 0.45 μm filter membrane.

8. The method according to claim 5, characterized in that After adding the solution or sample in the loading and elution steps, wait for 5 minutes and then use the gas supply device to generate a pressure difference for suction filtration.

9. The method according to claim 8, characterized in that The air supply device is a vacuum pump.

10. Use of the method according to claims 1 to 9 in purifying cell supernatant with high protein content.

Images