CN109369776B

CN109369776B - Protein purification system and method

Info

Publication number: CN109369776B
Application number: CN201811333500.1A
Authority: CN
Inventors: 徐志豪; 杨晓明; 汤炜; 张赶
Original assignee: Hangzhou Just Biotherapeutics Inc
Current assignee: Suzhou Chuangsheng Pharmaceutical Group Co ltd
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2020-12-25
Anticipated expiration: 2038-11-09
Also published as: CN109369776A

Abstract

The invention provides a protein purification system and a method. A protein purification system comprises a first sterilizing filter, a heat exchanger, a second sterilizing filter and a protein A affinity chromatographic column which are connected in sequence. The purification method comprises the following steps: the protein solution is subjected to a first filter sterilization, then heated, and then subjected to a second filter sterilization, followed by a protein a affinity chromatography treatment. The invention solves the problem that continuous purification can not be realized between low-temperature sterilization and normal-temperature affinity chromatography by adding two filters and adding a heat exchanger between the two filters, and improves the purification efficiency.

Description

Protein purification system and method

Technical Field

The invention relates to the field of protein purification, in particular to a system and a method for purifying protein.

Background

In recent years, the biopharmaceutical industry has been vigorously developed in China. The biological medicine has low toxic and side effect and high specific treatment effect, and brings good news to the majority of patients in China. However, the manufacturing process of biopharmaceuticals is still relatively backward and the productivity is low. One of the factors limiting productivity is that batch-wise production wastes significant production time. Compared with the prior art, the continuous production can greatly improve the production efficiency. Risks are often associated with the use of new production processes and capital investment is increased. Therefore, based on the existing production facilities and production modes, the continuous flow type production is realized by modifying, so that the risk can be reduced, and the cost of a company can be reduced. At the same time, it is easier to obtain the approval of national regulations.

In a continuous flow process for extended periods of time, the greatest challenge is to prevent microbial contamination of the protein product. Filtration and temperature reduction are among the effective ways to control microbial contamination. Chromatography can be continuously operated for a long time under the conditions of low temperature (no freezing) and sterile filtration (0.22 micron pore size) so as to achieve higher production efficiency, however, related equipment capable of continuously combining the low temperature, sterile filtration and chromatography does not exist at present.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a protein purification system, which solves the problem that continuous purification cannot be realized between low-temperature sterilization and normal-temperature affinity chromatography by adding two filters and adding a heat exchanger between the two filters, and improves the purification efficiency.

The second objective of the invention is to provide a protein purification method, which has the advantages of high efficiency, high yield, low cost, stability, reliability, little microbial pollution and the like compared with the traditional method.

In order to solve the technical problems, the invention provides the following technical scheme:

a protein purification system comprises a first sterilizing filter, a heat exchanger, a second sterilizing filter and a protein A affinity chromatographic column which are connected in sequence.

The working principle of the protein purification system is as follows:

the protein solution passes through the first sterilizing filter, is sterilized by filtration for one time, then passes through the heat exchanger, is slowly heated by the heat exchanger, the temperature of the protein solution reaches normal temperature (10-30 ℃) within certain buffering time, and then passes through the second sterilizing filter for sterilization by filtration, and then the protein solution can enter the protein A affinity chromatographic column for chromatographic treatment. In the process, the protein solution can enter the protein A affinity chromatography column through continuous treatment, so that the time of discontinuous operation is saved, the purification efficiency is improved, the working temperature of the protein A affinity chromatography is always in the optimal range, and the long-time continuous operation can be further realized.

On the other hand, the method adopts twice filtration sterilization, so that the content of bacteria and viruses in the protein solution is lower, and the problem of bubble precipitation caused by direct contact of the protein solution with a room-temperature environment at a low temperature is avoided by slowly heating the heat exchanger.

On the basis, the system can be further improved as follows.

Preferably, the pore diameter of the filter membrane in the first sterilizing filter is 0.20-0.22 μm.

Preferably, the pore diameter of the filter membrane in the second sterilizing filter is 0.20-0.22 μm.

Preferably, the heat exchanger is a water bath heater.

Preferably, the packing material of the protein a affinity chromatography column is MabSelect Sure, MabSelect LX, Amsphere A3, Praesto Jetted a50 or Prism a.

In the invention, the MabSelect LX, Amsphere A3, Praesto Jetted A50 and Prism A fillers are generally fixed manufacturers, namely GE Health Care company, JSR company, Purolite company and GE Health Care company respectively.

The protein purification method corresponding to the above purification system is as follows.

The protein solution is subjected to a first filter sterilization, then heated, and then subjected to a second filter sterilization, followed by a protein a affinity chromatography treatment.

Preferably, the method of protein a affinity chromatography is: after loading and before eluting the target antibody with an eluent, washing the column bed with an intermediate washing buffer;

the intermediate washing buffer solution is: a buffer solution with a pH value of 5-6 and a conductivity of 20-100 mS/cm.

The method comprises the steps of washing a sample by using a buffer solution with the pH value of 5-6 and the conductivity of 20-100 mS/cm, and removing HCP and DNA in the sample. Compared with a chromatography process without an intermediate washing step, the method has the advantages that the removal rate of HCP and DNA is obviously improved, the content of HCP in the obtained eluent is lower than 1000ppm, and the content of residual DNA is lower than 100 pg/mg.

The pH and conductivity of the intermediate wash buffer allow for removal of key parameters of HCP, DNA, the pH may take any value in the range of 5 to 6, such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, etc., and the conductivity may take any value in the range of 20 to 100mS/cm, such as 20mS/cm, 25mS/cm, 30mS/cm, 35mS/cm, 40mS/cm, 45mS/cm, 50mS/cm, 55mS/cm, 60mS/cm, 65mS/cm, 70mS/cm, 75mS/cm, 80mS/cm, 85mS/cm, 90mS/cm, 95mS/cm, 100mS/cm, etc.

On the basis of the addition of the intermediate cleaning, the following improvements can be made.

Preferably, the buffer is an acetate buffer system, a phosphate buffer system or a citrate buffer system.

The buffers do not introduce exogenous impurities, and the raw materials are easy to obtain.

The concentration of the buffer is not particularly limited, and may be, for example, 50mM NaAc-HAc or 1M NaAc-HAc.

Preferably, the buffer is a buffer for adjusting conductivity with a chloride salt, preferably sodium chloride.

Of course, other salts with good solubility can be used to adjust the conductivity, and the addition is not limited to the addition of one salt, or the addition of a plurality of salts in combination.

Through investigation, the effect of twice as much as half the effort can be achieved by adding the intermediate cleaning step in the chromatographic column filled with the filler, and the removal capability of HCP and DNA is obviously improved.

Preferably, the pH value of the intermediate washing buffer is 5.5-6, preferably 5.5-5.8.

Preferably, the conductivity is 50-100 mS/cm, preferably 50-80 mS/cm.

Preferably, the protein a affinity chromatography flow sequentially comprises: sterilizing, washing with an equilibrium solution, loading, washing with the equilibrium solution, washing with the intermediate washing buffer solution, washing with the equilibrium solution, and eluting the target antibody with the eluent.

Preferably, the equilibrium solution is Tris-HCl + 150-160 mM NaCl, pH 7.4-7.6 buffer solution, preferably 50-55 mM Tris-HCl.

Preferably, when the target antibody is a monoclonal antibody expressed by CHO cells, the eluent is NaAc-HAc, a buffer solution with a pH value of 3.0-3.5, and preferably 50-55 mM NaAc-HAc.

Preferably, the protein a affinity chromatography is followed by anion exchange chromatography of the collected eluate:

sequentially disinfecting, washing with balance liquid, loading and washing with balance liquid.

Preferably, the equilibrium solution during anion exchange chromatography is Tris-HCl and buffer solution with the pH value of 8.0-8.2.

Preferably, the filler for anion exchange chromatography is POROS 50HQ, Q Sepharose FF, Capto Q, or Eshmuno Q.

In summary, compared with the prior art, the invention achieves the following technical effects:

(1) by adding two filters and adding a heat exchanger between the two filters, the problem that continuous purification cannot be realized between low-temperature sterilization and normal-temperature affinity chromatography is solved, and the purification efficiency is improved;

(2) by adding an intermediate washing step when the antibody is purified by protein A affinity chromatography, the removal capacity of HCP and DNA is improved, and compared with the traditional chromatography, the method is substantially improved;

(3) the capability of the purification process for removing HCP and DNA is further improved by optimizing other process parameters in protein A affinity chromatography and anion exchange chromatography.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of a protein purification system provided by the present invention;

reference numerals:

1-a first sterilizing filter, 2-a heat exchanger, 3-a second sterilizing filter and 4-a protein affinity chromatographic column.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following detailed description, but those skilled in the art will understand that the following described examples are some, not all, of the examples of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

A protein purification system is shown in figure 1, wherein the arrows indicate the flow direction of protein solution, and comprises a first sterilizing filter 1 (with a membrane pore size of 0.22 μm), a heat exchanger 2, a second sterilizing filter 3 (with a membrane pore size of 0.22 μm), and a protein A affinity chromatography column 4 connected in sequence.

Using a packed Prism A (GE), a chromatography column Econoline 5/250(Essential life), a chromatography column having a diameter of 0.5cm, a column height of 20.5cm and a column volume of 4ml as an example, the flow rate was 300 cm/h.

The sample source was monoclonal antibody expressed by CHO cells (Mab 1). Sequentially passes through a first sterilizing filter, a heat exchanger and a second sterilizing filter, wherein the heat exchanger heats the protein solution to 25 ℃, and then the protein solution enters a chromatographic column after sterilizing and filtering.

The purification process in the chromatographic column comprises the following steps: washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution 50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) onto a chromatography column; washing the bed with 3CV of equilibration solution, washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc +1M NaCl, pH5.5), and washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc, pH 5.5); eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; the column was washed with 3CV of 1mol/L acetic acid solution and equilibrated with 2CV of equilibration solution. The cycle was continued for 14 days. The HCP impurity content in the resulting eluate was 565 ppm.

Example 2

A protein purification system is shown in figure 1, wherein the arrows indicate the flow direction of protein solution, and comprises a first sterilizing filter 1 with a membrane pore size of 0.22 μm, a heat exchanger 2, a second sterilizing filter 3 with a membrane pore size of 0.22 μm), and a protein A affinity chromatography column 4

Using the packing Praesto Jetted A50(Purolite), the chromatography column Vantage 11/250(Millipore), a chromatography column having a diameter of 1.1cm, a column height of 20cm and a column volume of 20ml as an example, the flow rate was 300 cm/h.

The sample source was monoclonal antibody expressed by CHO cells (Mab 2). Sequentially passes through a first sterilizing filter, a heat exchanger and a second sterilizing filter, wherein the heat exchanger heats the protein solution to 25 ℃, and then the protein solution enters a chromatographic column after sterilizing and filtering.

The purification process in the chromatographic column comprises the following steps: washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution 50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) onto a chromatography column; washing the bed with 3CV of equilibration solution, washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc +1M NaCl, pH5.5), and washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc, pH 5.5); eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; the column was washed with 3CV of 1mol/L acetic acid solution and equilibrated with 2CV of equilibration solution. The cycle was continued for 14 days. The HCP impurity content in the resulting eluate was 835ppm

Comparative example 1

The protein purification system is a conventional set-up. The product is directly connected with a chromatography system without temperature control and sterile filtration.

The sample source was monoclonal antibody expressed by CHO cells (Mab 1). Is subjected to deep filtration and clarification treatment.

The purification process in the chromatographic column comprises the following steps: washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution 50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) onto a chromatography column; washing the bed with 3CV of equilibration solution, washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc +1M NaCl, pH5.5), and washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc, pH 5.5); eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; the column was washed with 3CV of 1mol/L acetic acid solution and equilibrated with 2CV of equilibration solution. After circulating for 2 days, the chromatographic column obviously grows bacteria. The chromatographic column changes color and the column pressure rises. The chromatography experiment was terminated. The HCP impurity content in the resulting eluate was 1288 ppm.

Comparative example 2

The sample source was monoclonal antibody expressed by CHO cells (Mab 2). Is subjected to deep filtration and clarification treatment. Sequentially cooling, sterilizing and filtering to enter a chromatographic column.

The purification process in the chromatographic column comprises the following steps: washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution 50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) onto a chromatography column; washing the bed with 3CV of equilibration solution, washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc +1M NaCl, pH5.5), and washing the bed with 3CV of intermediate wash buffer (50mM NaAc-HAc, pH 5.5); eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; the column was washed with 3CV of 1mol/L acetic acid solution and equilibrated with 2CV of equilibration solution. After 1 hour of circulation, bubbles were generated in the chromatography system and column (as the cold liquid encountered the system and column at room temperature, air evolved). The chromatography experiment was terminated. The HCP impurity content in the eluate obtained was 753 ppm.

Comparison of the results of examples 1, 2 and comparative examples 1, 2:

comparing examples 1 and 2 with comparative examples 1 and 2, it was found that the use of the filtration heat exchange process enables the chromatography system to operate efficiently for a long period of time for 14 days, whereas the chromatography system without the apparatus cannot operate stably for a long period of time (at most 2 days) because of bacterial growth in the chromatography system or because of bubble evolution. In addition, the removal of impurities (HCP) is not affected by the use of a new device. The effect of chromatography to remove HCP was rather better.

Example 3

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0mL, operating flow 220 cm/h. The sample source was Mab expressed by CHO cells (accession number Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (50mM NaAc-HAc +1M NaCl, pH5.5, conductivity 88.5mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 900ppm, and the DNA-remaining amount was 85 pg/mg.

POROS 50HQ, chromatography column BenchMark 06/25(Omnifit), diameter 0.66cm, column height 20cm, column volume 6.8mL, operating flow 220 cm/h. The sample source was the affinity eluate from the previous step, adjusted to pH7.5 and filtered through a 0.22 μm filter with an X-MuLV addition of 1% (v/v). Washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution (50mM Tris-HCl, pH 7.5); loading the sample after pH adjustment and clarification to a chromatographic column, wherein the loading capacity is 100 g/L; washing the column bed with 3CV of balance liquid, and collecting flow-through liquid; finally, regenerating the chromatographic column by using a regeneration solution (50mM Tris-HCl +1M NaCl, pH7.5); washing the column with 3CV of 0.5mol/L sodium hydroxide solution, and preserving the column with 3CV of preserving solution. The cleaning capacity of the step on X-MuLV can reach 5.1log 10.

Comparative example 3

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0mL, operating flow 220 cm/h. The sample source was Mab expressed by CHO cells (accession number Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of balancing liquid; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 3600ppm, and the DNA residue was 400 pg/mg.

POROS 50HQ, chromatography column BenchMark 06/25(Omnifit), diameter 0.66cm, column height 20cm, column volume 6.8mL, operating flow 220 cm/h. The sample source was the affinity eluate from the previous step, adjusted to pH7.5 and filtered through a 0.22 μm filter with an X-MuLV addition of 1% (v/v). Washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution (50mM Tris-HCl, pH 7.5); loading the sample after pH adjustment and clarification to a chromatographic column, wherein the loading capacity is 100 g/L; washing the column bed with 3CV of balance liquid, and collecting flow-through liquid; finally, regenerating the chromatographic column by using a regeneration solution (50mM Tris-HCl +1M NaCl, pH7.5); washing the column with 3CV of 0.5mol/L sodium hydroxide solution, and preserving the column with 3CV of preserving solution. The clearance capacity of this intermediate washing step without affinity chromatography was 4.0log 10.

Comparing example 3 with comparative example 3, it can be seen that: the affinity intermediate washing step can effectively improve the cleaning capacity of the X-MuLV.

Example 4

MabSelect SuRe, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0ml, operating flow rate 200 cm/h. The sample source was monoclonal antibody expressed by CHO cells (Mab 2). Washing the column with 3CV of 0.1mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (20mM Tris-HCl +1M NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 30 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (1M NaAc-HAc, pH5.8, conductivity 47.3mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (100mM HAc, pH3.0), and collecting eluate; finally, the chromatographic column is washed by 3CV of 0.1mol/L sodium hydroxide solution, and after the chromatographic column is balanced by 3CV of balancing solution, the chromatographic column is finally preserved by 3CV of preserving solution. The HCP content of the eluate was 960ppm, and the residual amount of DNA was 90 pg/mg.

Q Sepharose FF, column BenchMark 06/25(Omnifit), diameter 0.66cm, column height 20cm, column volume 6.8ml, operating flow 200 cm/h. The sample source was the affinity eluent from the previous step, adjusted to pH8.0 and filtered through a 0.22 μm filter with an added amount of MVM of 1% (v/v). Washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution (50mM Tris-HCl, pH 8.0); loading the sample after pH adjustment and clarification to a chromatographic column, wherein the loading capacity is 60 g/L; washing the column bed with 3CV of balance liquid, and collecting flow-through liquid; finally, regenerating the chromatographic column by using a regeneration solution (50mM Tris-HCl +1M NaCl, pH8.0); washing the column with 3CV of 0.5mol/L sodium hydroxide solution, and preserving the column with 3CV of preserving solution. This step may achieve a 4.5log10 clearance of MVM.

Comparative example 4

MabSelect SuRe, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0ml, operating flow rate 200 cm/h. The sample source was monoclonal antibody expressed by CHO cells (Mab 2). Washing the column with 3CV of 0.1mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (20mM Tris-HCl +1M NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 30 g/L; cleaning the column bed by using 3CV balancing liquid, and cleaning the column bed by using 3CV balancing liquid; eluting with 3.5CV of eluent (100mM HAc, pH3.0), and collecting eluate; finally, the chromatographic column is washed by 3CV of 0.1mol/L sodium hydroxide solution, and after the chromatographic column is balanced by 3CV of balancing solution, the chromatographic column is finally preserved by 3CV of preserving solution. The HCP content of the eluate was 3710ppm, and the residual amount of DNA was 524 pg/mg.

Q Sepharose FF, column BenchMark 06/25(Omnifit), diameter 0.66cm, column height 20cm, column volume 6.8ml, operating flow 200 cm/h. The sample source was the affinity eluent from the previous step, adjusted to pH8.0 and filtered through a 0.22 μm filter with an added amount of MVM of 1% (v/v). Washing the column with 3CV of 0.5mol/L NaOH solution, and then balancing the column with 3CV of balancing solution (50mM Tris-HCl, pH 8.0); loading the sample after pH adjustment and clarification to a chromatographic column, wherein the loading capacity is 60 g/L; washing the column bed with 3CV of balance liquid, and collecting flow-through liquid; finally, regenerating the chromatographic column by using a regeneration solution (50mM Tris-HCl +1M NaCl, pH8.0); washing the column with 3CV of 0.5mol/L sodium hydroxide solution, and preserving the column with 3CV of preserving solution. The clearance capacity of the intermediate wash step without affinity chromatography was 3.3log 10.

Comparing example 4 with comparative example 4, it can be seen that: the affinity intermediate cleaning step can effectively improve the cleaning capability of the MVM.

Example 5

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0ml, operating flow 220 cm/h. The sample source was monoclonal antibody expressed by CHO cells (Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (50mM NaAc-HAc +1M NaCl, pH5.0, conductivity 91.3mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 810ppm, and the residual amount of DNA was 78 pg/mg.

Example 6

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0ml, operating flow 220 cm/h. The sample source was monoclonal antibody expressed by CHO cells (Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (1M NaAc-HAc + NaCl, pH6.0, conductivity 85.1mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 970ppm, and the residual amount of DNA was 90 pg/mg.

Example 7

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0mL, operating flow 220 cm/h. The sample source was Mab expressed by CHO cells (accession number Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (50mM NaAc-HAc +0.2M NaCl, pH5.5, conductivity 20mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 990ppm, and the residual amount of DNA was 98 pg/mg.

Example 8

MabSelect SuRe LX, chromatography column Vantage 11/250(Millipore), diameter 1.1cm, column height 20cm, column volume 19.0mL, operating flow 220 cm/h. The sample source was Mab expressed by CHO cells (accession number Mab 1). Washing the column with 3CV of 0.2mol/L NaOH solution, and then balancing the column with 5CV of balancing solution (50mM Tris-HCl +150mM NaCl, pH 7.4); loading the clarified cell culture Harvest (HCCF) to a chromatographic column, wherein the loading capacity is 50 g/L; washing the column bed with 3CV of equilibration solution, washing the column bed with 3CV of intermediate washing buffer (50mM NaAc-HAc +1.2M NaCl, pH5.5, conductivity 100mS/cm), and washing the column bed with 3CV of equilibration solution; eluting with 3.5CV of eluent (50mM NaAc-HAc, pH3.5), and collecting eluate; and finally, washing the chromatographic column by using 3CV of 0.2mol/L sodium hydroxide solution, balancing the chromatographic column by using 3CV of balancing solution, and finally preserving the chromatographic column by using 3CV of preserving solution. The HCP content of the eluate was 890ppm, and the residual amount of DNA was 90 pg/mg.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein purification system is characterized by consisting of a first sterilizing filter, a heat exchanger, a second sterilizing filter and a protein A affinity chromatographic column which are connected in sequence;

the aperture of a filter membrane in the first sterilizing filter is 0.20-0.22 mu m;

the aperture of the filter membrane in the second sterilizing filter is 0.20-0.22 mu m;

the filler of the protein A affinity chromatographic column is MabSelect Sure, MabSelect LX, AmsphereA3, Praesto Jetted A50 or Prism A;

the purification system is used for continuous purification of proteins.

2. The protein purification system according to claim 1, wherein the heat exchanger is a water bath heater.

3. A method for purifying a protein, comprising the steps of:

passing a protein solution through the protein purification system of claim 1;

the heat exchanger heats the protein solution;

the flow of the protein A affinity chromatography sequentially comprises the following steps: sterilizing, washing with a balance solution, loading, washing with the balance solution, washing with an intermediate washing buffer solution, washing with the balance solution and eluting a target antibody with an eluent;

the intermediate washing buffer solution is: a buffer solution with a pH value of 5-6 and a conductivity of 20-100 mS/cm;

the intermediate washing buffer solution is an acetate buffer system, a phosphate buffer system or a citrate buffer system;

the intermediate wash buffer was conductivity adjusted using chloride salts.

4. The protein purification method according to claim 3, wherein the heating temperature is 10 to 30 ℃.

5. The protein purification method according to claim 4, wherein the heating temperature is 10 to 25 ℃.

6. The protein purification method according to claim 3, wherein the chloride salt is sodium chloride.

7. The protein purification method according to claim 3, wherein the pH value of the intermediate washing buffer is 5.5 to 6.

8. The protein purification method according to claim 7, wherein the pH value of the intermediate washing buffer is 5.5 to 5.8.

9. The protein purification method according to claim 3, wherein the conductivity of the intermediate wash buffer is 50-100 mS/cm.

10. The protein purification method according to claim 9, wherein the conductivity of the intermediate wash buffer is 50-80 mS/cm.

11. The protein purification method according to any one of claims 3 to 10, wherein the equilibration solution is Tris-HCl +150 to 160mM NaCl, pH7.4 to 7.6 buffer solution.

12. The protein purification method according to claim 11, wherein the equilibrium solution is 50 to 55mM Tris-HCl.

13. The method for protein purification according to claim 11, wherein when the target antibody is a monoclonal antibody expressed by CHO cells, the elution solution is NaAc-HAc buffer solution having a pH of 3.0 to 3.5.

14. The protein purification method according to claim 13, wherein the eluent is 50 to 55mM NaAc-HAc.

15. The protein purification method according to claim 3, further comprising subjecting the collected eluate to anion exchange chromatography after the protein A affinity chromatography:

and sequentially sterilizing, washing with a balance solution, loading and washing with the balance solution.

16. The method for purifying protein according to claim 15, wherein the equilibrium solution for anion exchange chromatography is Tris-HCl, pH 8.0-8.2 buffer solution.

17. The protein purification process according to claim 16, wherein the filler for anion exchange chromatography is POROS 50HQ, Q Sepharose FF, Capto Q, or Eshmuno Q.