CN111440226B - System and method for protein purification - Google Patents
System and method for protein purification Download PDFInfo
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- CN111440226B CN111440226B CN202010460856.2A CN202010460856A CN111440226B CN 111440226 B CN111440226 B CN 111440226B CN 202010460856 A CN202010460856 A CN 202010460856A CN 111440226 B CN111440226 B CN 111440226B
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention relates to the technical field of protein purification, in particular to a system and a method for protein purification. A system for protein purification, comprising a depth filtration unit, a chromatography unit and a peristaltic pump; the outlet of a peristaltic pump pipe of the peristaltic pump is connected with the inlet of the deep layer filtering unit; the outlet of the depth filtration unit is connected to the inlet of the chromatography unit; the chromatography unit comprises a chromatographic membrane and/or a chromatographic column; the depth filtration unit comprises a depth filtration membrane; the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2Fraction (100-110) mL; the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2And (0.8-10) L. According to the invention, the deep filtration membrane is matched with the chromatographic membrane and/or the chromatographic column, so that multiple steps can be processed simultaneously, and the production efficiency is greatly improved; in addition, the existing full-automatic chromatography system is not needed, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of protein purification, in particular to a system and a method for protein purification.
Background
The large-scale economic purification of proteins is becoming an increasingly important issue for the biotechnology industry. Production costs are becoming an important factor in protein purification. The traditional antibody drug purification method in the past generally comprises antibody affinity capture, virus inactivation, intermediate depth filtration, ion exchange chromatography, concentration and percolation. In conventional production, the apparatus for depth filtration usually consists of a peristaltic pump and a pressure gauge, while the apparatus for chromatography is a fully automated chromatography system. Chromatography systems are often expensive to operate at one time and cannot handle multiple steps simultaneously, which results in separate operations for depth filtration and chromatography, which greatly reduces production efficiency and increases production costs.
Chromatography is widely used in the industry because of its excellent separation and economy, and common chromatography modes include bind-elute mode chromatography and flow-through mode chromatography. The downstream purification platform processes currently used by many companies are typically a combination of anion exchange chromatography in flow-through mode and cation exchange chromatography in combination with elution mode to remove process-related and product-related impurities. The flow-through mode allows the antibody to pass through the chromatography column directly while binding impurities to the packing, resulting in a higher loading of the flow-through mode and fewer washing and elution steps, thereby increasing productivity and reducing production costs, compared to the binding-elution mode.
However, current protein purification involves multiple steps, such as multiple depth filtration, multiple chromatography. However, the steps have the defects of zero operation fragmentation, long time, large equipment investment, low production efficiency and the like in the actual amplification production.
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 technical problems of low production efficiency and large equipment investment in the prior art.
The second object of the present invention is to provide a method for protein purification, which can integrate a plurality of operation steps, and has high production efficiency and low equipment investment.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a system for protein purification, comprising a depth filtration unit, a chromatography unit and a peristaltic pump;
the outlet of a peristaltic pump pipe of the peristaltic pump is connected with the inlet of the deep layer filtering unit; the outlet of the depth filtration unit is connected to the inlet of the chromatography unit;
the chromatography unit comprises a chromatographic membrane and/or a chromatographic column; the depth filtration unit comprises a depth filtration membrane;
the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2﹕(100～110)mL；
The ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2﹕(0.8～10)L。
According to the invention, the deep filtration membrane is matched with the chromatographic membrane and/or the chromatographic column, so that multiple steps can be processed simultaneously, the deep filtration treatment and the chromatographic treatment do not need to be operated separately, and the production efficiency is greatly improved; in addition, the existing full-automatic chromatography system is not needed, and the production cost is reduced.
In addition, the invention adopts the corresponding membrane area and the membrane volume of the chromatographic membrane or the column volume of the chromatographic column, can ensure that the chromatographic treatment is directly carried out after the deep filtration, and the condition of overhigh pressure of a single step can not occur.
The protein purification system of the invention reduces time cost and improves production efficiency while using a chromatography system.
In a preferred embodiment of the invention, the system comprises a peristaltic pump. The protein purification system of the invention can integrate the deep filtration treatment with one step of chromatography step or multiple steps of chromatography step only by one peristaltic pump, and adopts appropriate corresponding membrane area and membrane volume of chromatography membrane or column volume of chromatography column, so that the pressure of each operation step can not be too high.
The inlet of the peristaltic pump tube of the peristaltic pump is externally connected with water, a balance buffer solution or a sample and the like, and the adjustment is carried out according to the actual processing operation stage.
In a specific embodiment of the present invention, the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2Fraction (102 to 108) mL, preferably 1m2(104 to 105) mL, more preferably 1.1m2﹕115mL。
In a specific embodiment of the present invention, when the chromatography unit is a chromatography column, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2Ratio (8.5 to 9.5) L, more preferably 1.1m2﹕9.8L。
In a specific embodiment of the present invention, when the chromatography unit comprises a chromatography column and a chromatography membrane, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2(0.8 to 1) L, more preferably 1.1m2﹕1L。
In a particular embodiment of the invention, the chromatography unit comprises one chromatography membrane or a plurality of chromatography membranes connected in series;
alternatively, the chromatography unit comprises one chromatography column or a plurality of chromatography columns connected in series;
or, the chromatography unit comprises a chromatography membrane and a chromatography column which are connected in series;
or the chromatography unit comprises a chromatographic membrane and a plurality of chromatographic columns connected in series, and the chromatographic membrane and the plurality of chromatographic columns connected in series are connected in series;
or the chromatography unit comprises a plurality of chromatographic membranes and a chromatographic column which are connected in series, wherein the plurality of chromatographic membranes and the chromatographic column are connected in series.
The number of the chromatographic columns and the chromatographic membranes can be adjusted and selected according to the actual operation requirement.
In a preferred embodiment of the invention, the system further comprises at least one pressure gauge. The operating pressure of each step is monitored by a pressure gauge, so that overhigh pressure is avoided; according to the pressure condition fed back by the pressure gauge, when the pressure is too high, the flow rate can be properly reduced by the peristaltic pump, so that the pressure is in a controllable range.
In a preferred embodiment of the invention, the pressure gauge is arranged between the depth filtration unit and the chromatography unit.
In a preferred embodiment of the present invention, the pressure gauge is disposed between the peristaltic pump and the depth filtration unit.
In a specific embodiment of the invention, pressure gauges are arranged between chromatographic membranes, between chromatographic columns and between chromatographic membranes and chromatographic columns of the chromatography unit.
In a specific embodiment of the invention, the chromatographic membrane comprises an anion exchange chromatographic membrane. Preferably, the anion exchange chromatography membrane is
HD-Q anion exchange chromatography membrane.
In a specific embodiment of the present invention, the chromatography column comprises any one of a mixed-mode chromatography column, an anion-exchange chromatography column and a cation-exchange chromatography columnOne or more of them. Preferably, the chromatographic column is a Capto Adhere ImpRes chromatographic column or
CP-FT chromatographic column.
The Capto Adhere ImpRes chromatographic column refers to a Capto Adhere ImpRes filling material;
the CP-FT chromatographic column is filled with
CP-FT。
In a specific embodiment of the present invention, the depth filtration membrane is any one of A1HC depth filtration membrane, a C0SP depth filtration membrane and an X0HC depth filtration membrane.
The invention also provides a method for purifying protein by using any one of the protein purification systems, which comprises the following steps:
after a sample to be purified is loaded by a peristaltic pump, carrying out deep filtration treatment and chromatography treatment by the deep filtration unit and the chromatography unit respectively; the chromatography is flow-through mode chromatography.
The present invention integrates depth filtration and flow-through mode chromatography steps therein such that both depth filtration and chromatography steps are performed simultaneously using only peristaltic pumps and pressure gauges. The time cost and the use of the chromatography system are reduced, and the production efficiency is improved.
After being subjected to a depth filtration treatment by a depth filtration unit, the sample is directly loaded onto the chromatography unit, such as a chromatographic membrane and/or a chromatographic column.
In a specific embodiment of the invention, the flow-through mode chromatography is a one-step flow-through mode chromatography or a multi-step flow-through mode chromatography.
In a specific embodiment of the present invention, the flow rate of the depth filtration treatment is 50 to 200LMH, and the flow rate of the chromatography treatment is 20 to 50mL/min or 200 to 300 cm/hr.
In a specific embodiment of the invention, the flow rate is adjusted so that the pressure between the depth filtration treatment and the chromatography treatment before the depth filtration treatment is 0-2 Bar, preferably > 0Bar and < 1 Bar.
In practice, the pressure is the pressure caused by the flow of the sample before the depth filtration process and between the depth filtration process and the chromatography process.
In a specific embodiment of the invention, the loading amount of the chromatography unit is 1000-2000 g/m2Or 200-400 g/L.
In a specific embodiment of the present invention, the sample to be purified comprises any one of a monoclonal antibody and an Fc fusion protein.
Further, the pH of the sample of the monoclonal antibody is 7.0-7.6, and the conductivity of the sample is 5-10 mS/cm; the pH value of the Fc fusion protein sample is 5.5-7.6, and the conductivity is less than 5 mS/cm.
In a specific embodiment of the present invention, the depth filtration unit is subjected to a water washing treatment before the depth filtration treatment.
Specifically, in the water washing treatment, the ratio of the volume of water to the membrane area of the deep filtration membrane is (95-105) L: 1m2Preferably 100L: 1m2。
In a particular embodiment of the invention, the chromatography unit is sterilized before the depth filtration process.
In a particular embodiment of the invention, the sterilization treatment comprises an alkaline cleaning sterilization treatment. Further, the alkali washing sterilization treatment is performed by using an aqueous NaOH solution. Specifically, the concentration of the NaOH aqueous solution can be 0.1-1 mol/L, and preferably 0.5 mol/L.
In a specific embodiment of the present invention, the depth filtration unit subjected to the water washing process and the chromatography unit subjected to the sterilization process are equilibrated with an equilibration buffer before the depth filtration process.
Specifically, the balancing process includes: balancing the depth filtration unit and the chromatography unit with an equilibration buffer, the depth filtration unit having an equilibration flux of 15 to E60L/m2And balancing the chromatography units for 3-5 CV. Wherein, when the chromatography unit is a chromatography column, CV refers to the column volume; when the chromatography unit is a chromatography membrane, CV represents the volume of the chromatography membrane.
In practice, after the water washing treatment of the depth filtration unit and the disinfection treatment of the chromatography unit, the outlet of the depth filtration unit is connected to the inlet of the chromatography unit, and then the depth filtration unit and the chromatography unit are equilibrated with an equilibration buffer.
Specifically, the equilibrium buffer is adjusted according to the types of the different depth filtration units and the chromatography units.
As in the specific embodiment, when the depth filtration unit is an A1HC depth filtration membrane, the chromatography unit is
In the case of HD-Q chromatography, the pH of the equilibration buffer is 7.5, specifically 50mM Tris-HAc; the balance flux of the deep filtration unit is 15-25L/m2Preferably 20 to 25L/m2. When the depth filtration unit is a C0SP depth filtration membrane and the chromatography unit is a Capto Adhere ImpRes chromatography column, the pH of the equilibration buffer is 7.0, the conductivity is 10mS/cm, specifically 50mM Tris-HAc, 100mM NaCl; the balance flux of the deep filtration unit is 40-50L/m2Preferably 45 to 50L/m2. When the deep filtration unit is an X0HC deep filtration membrane, the chromatography units are connected in series
HD-Q chromatographic carrier and
in the case of CP-FT column chromatography, the pH of the equilibration buffer is 5.5, specifically 50mM NaAc-HAc; the balance flux of the deep filtration unit is 50-60L/m2Preferably 54 to 60L/m2。
In a specific embodiment of the present invention, after the loading is finished, the top washing treatment is performed on the depth filtration unit and the chromatography unit by using the equilibration buffer.
Starting the process from the loading, collecting the flow-through solution at the end outlet of the chromatography unit until the collection is completed after the top-washing process.
In practice, after collection is completed, the deep filtration unit and the chromatography unit are disconnected, the deep filtration unit, such as a deep filtration membrane, can be discarded, and the chromatography unit is regenerated and then preserved with a sufficient amount of preservation solution. The operation of the specific regeneration treatment can be adjusted according to different chromatographic columns and/or chromatographic membranes.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the invention, the deep filtration membrane is matched with the chromatographic membrane and/or the chromatographic column, so that multiple steps can be processed simultaneously, the deep filtration treatment and the chromatographic treatment do not need to be operated separately, and the production efficiency is greatly improved; in addition, the existing full-automatic chromatography system is not needed, so that the production cost is reduced;
(2) the invention adopts corresponding membrane area and membrane volume of chromatographic membrane or column volume of chromatographic column, which can ensure that the chromatographic treatment can be directly carried out after deep filtration without the condition of overhigh pressure of single step;
(3) the invention integrates the steps of deep filtration and flow-through mode chromatography, so that the steps of deep filtration and chromatography are simultaneously carried out only by devices such as a peristaltic pump, a pressure gauge and the like; the production efficiency is improved while the time cost and the use of the chromatography system are reduced.
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.
This example provides a protein purification system comprising a depth filtration unit, a chromatography unit, and a peristaltic pump; the outlet of a peristaltic pump pipe of the peristaltic pump is connected with the inlet of the deep layer filtering unit; the outlet of the depth filtration unit is connected to the inlet of the chromatography unit;
the chromatography unit comprises a chromatography membrane and/or a chromatography column; the depth filtration unit comprises a depth filtration membrane;
the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2﹕(100～110)mL；
The ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2﹕(8～10)L。
In a preferred embodiment of the invention, the system comprises a peristaltic pump. The protein purification system of the invention can integrate the deep filtration treatment with one step of chromatography step or multiple steps of chromatography step only by one peristaltic pump, and adopts appropriate corresponding membrane area and membrane volume of chromatography membrane or column volume of chromatography column, so that the pressure of each operation step can not be too high.
In a specific embodiment of the present invention, the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2Fraction (102 to 108) mL, preferably 1m2(104 to 105) mL, more preferably 1.1m2﹕115mL。
As in the different embodiments, the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatography membrane may be 1m2﹕102mL、1m2﹕103mL、1m2﹕104mL、1m2﹕105mL、1m2﹕106mL、1m2﹕107mL、1m2And (6) 108mL, and selection can be carried out according to actual requirements. Optimized to 1.1m2And 115mL, the best stability. The above ratio refers to the individual deep layersThe ratio of the membrane area of the filtration membrane to the membrane volume of the individual chromatographic membranes.
In an embodiment of the present invention, a ratio of a membrane area of the deep filtration membrane to a column volume of the chromatography column is 1m2Ratio (8.5 to 9.5) L, more preferably 1.1m2﹕9.8L。
As in the different embodiments, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column may be 1m2﹕8.5L、1m2﹕8.6L、1m2﹕8.7L、1m2﹕8.8L、1m2﹕8.9L、1m2﹕9.0L、1m2﹕9.1L、1m2﹕9.2L、1m2﹕9.3L、1m2﹕9.4L、1m2And a ratio of 9.5L, and the like, and can be selected according to actual requirements. Optimized to 1.1m2The stability was best at a ratio of 9.8L. The above ratio refers to the ratio of the membrane area of a single deep filtration membrane to the column volume of a single chromatography column.
In a particular embodiment of the invention, the chromatography unit comprises one chromatography membrane or a plurality of chromatography membranes connected in series;
alternatively, the chromatography unit comprises one chromatography column or a plurality of chromatography columns connected in series;
or, the chromatography unit comprises a chromatography membrane and a chromatography column which are connected in series;
or the chromatography unit comprises a chromatographic membrane and a plurality of chromatographic columns connected in series, and the chromatographic membrane and the plurality of chromatographic columns connected in series are connected in series;
or the chromatography unit comprises a plurality of chromatographic membranes and a chromatographic column which are connected in series, wherein the plurality of chromatographic membranes and the chromatographic column are connected in series.
The number of the chromatographic columns and the chromatographic membranes can be adjusted and selected according to the actual operation requirement.
In a preferred embodiment of the invention, the system further comprises at least one pressure gauge. The operating pressure of each step is monitored by a pressure gauge, so that overhigh pressure is avoided; according to the pressure condition fed back by the pressure gauge, when the pressure is too high, the flow rate can be properly reduced by the peristaltic pump, so that the pressure is in a controllable range.
In a preferred embodiment of the invention, the pressure gauge is arranged between the depth filtration unit and the chromatography unit.
In a specific embodiment of the invention, pressure gauges are arranged between chromatographic membranes, between chromatographic columns and between chromatographic columns of the chromatographic unit and between chromatographic membranes and chromatographic columns to monitor the pressure before each chromatographic membrane or chromatographic column is loaded.
In a specific embodiment of the invention, the chromatographic membrane comprises an anion exchange chromatographic membrane. Preferably, the anion exchange chromatography membrane is
HD-Q anion exchange chromatography membrane.
In a specific embodiment of the invention, the chromatography column comprises any one or more of a mixed mode chromatography column, an anion exchange chromatography column and a cation exchange chromatography column. Preferably, the chromatographic column is a Capto Adhere ImpRes chromatographic column or
CP-FT chromatographic column.
The Capto Adhere ImpRes chromatographic column refers to a Capto Adhere ImpRes filling material;
the CP-FT chromatographic column is filled with
CP-FT。
In a specific embodiment of the present invention, the depth filtration membrane is any one of A1HC depth filtration membrane, a C0SP depth filtration membrane, and an X0HC depth filtration membrane.
Example 1
The protein purification system of this example, which integrates depth filtration with a flow-through mode anion exchange chromatography, comprises a depth filtration membrane, an anion exchange chromatography membrane, and a peristaltic pump; the outlet of the peristaltic pipe of the peristaltic pump is connected to the inlet of the deep filtration membrane, and the outlet of the deep filtration membrane is connected to the inlet of the anion exchange chromatography membrane. Pressure gauges are arranged between the peristaltic pump and the deep filtration membrane and between the deep filtration membrane and the anion exchange chromatography membrane.
Specifically, the deep filtration membrane is A1HC deep filtration membrane of Merck, and the anion exchange chromatography membrane is
HD-Q anion exchange chromatography membrane.
This example provides a method of protein purification comprising the steps of:
(1) using 1.1m2A1HC depth filtration Membrane of (1) and 115mL
The HD-Q anion exchange chromatographic membrane is prepared by washing the deep filtration membrane with 110L pure water, washing the anion exchange chromatographic membrane with 0.5M NaOH aqueous solution to sterilize, and mixing the outlet of A1HC deep filtration membrane with the filtrate of the filtrate A and the filtrate of filtrate A1L
The inlet of the HD-Q anion exchange chromatography membrane is connected.
(2) A1HC submerged filter membrane equilibrated in well-ligated pH 7.5 with more than 22L of 50mM Tris-HAc and
and (3) carrying out a sample loading operation after the balance of the HD-Q anion exchange chromatographic membrane is completed.
(3) The sample is a monoclonal antibody, and the conditions of loading the sample are that the pH is 7.5 and the conductivity is less than 6 mS/cm; loading the sample to a deep filtration membrane at a flow rate of 200LMH by a peristaltic pump, and directly loading the sample to a A1HC deep filtration membrane
An HD-Q anion exchange chromatography membrane; monitoring of depth filtration membranes and anion exchange during loadingKeeping the pressure in the range of 0-2 Bar before the chromatographic membrane, and properly reducing the flow rate to about 50LMH when the pressure rises to exceed the range so as to restore the pressure to be in the range;
collecting sample flow-through liquid at the beginning of sample loading, namely at the outlet end of the anion exchange chromatographic membrane, wherein the sample loading amount reaches 1000g/m2After that, the sample application is ended.
(4) After the loading was complete, A1HC deep filtration membrane and the equilibration buffer were filtered through a peristaltic pump using more than 33L of the equilibration buffer
And (3) washing out the residual sample in the HD-Q anion exchange chromatographic membrane from the top, and finally harvesting the flow-through sample subjected to the deep filtration treatment and the anion exchange chromatography treatment.
(5) After the collection is completed, the connection between the deep filtration membrane and the anion exchange chromatography membrane is disconnected, the deep filtration membrane is discarded, then the used anion exchange chromatography membrane is disinfected and regenerated by using 0.5M NaOH aqueous solution, and then the disinfected and regenerated anion exchange chromatography membrane is preserved by using preservation solution for subsequent purification.
The results are shown in Table 1. The control group adopts a common purification process, the deep filtration and the anion exchange chromatography are carried out step by step, and the experimental group adopts a continuous flow process. By comparison with the conventional process, the productivity of this embodiment is improved by nearly 50%, and the product quality is kept consistent.
Table 1 example 1 experimental results
Example 2
The protein purification system of this example, which integrates depth filtration with a flow-through mode mixed mode chromatography column, comprises a depth filtration membrane, a mixed mode chromatography column, and a peristaltic pump; the outlet of the peristaltic pipe of the peristaltic pump is connected to the inlet of the deep filtration membrane, and the outlet of the deep filtration membrane is connected to the inlet of the mixed-mode chromatography column. Pressure gauges are arranged between the peristaltic pump and the deep filtration membrane and between the deep filtration membrane and the mixed mode chromatographic column.
Specifically, the deep filtration membrane is a C0SP deep filtration membrane of Merck, the mixed-mode chromatography column is a Capto Adhere Impres chromatography column, and the filler is Capto Adhere Impres of GE.
This example provides a method of protein purification comprising the steps of:
(1) using 1.1m2The C0SP deep filtration membrane and a 9.8L Capto Adhere Impres column (diameter 25cm, column height 20cm) were washed with 110L pure water, and the Capto Adhere Impres column was sterilized with 0.5M NaOH solution in an alkaline solution, and after washing and sterilization, the outlet of the C0SP deep filtration membrane was connected to the inlet of the Capto Adhere Impres column.
(2) The well-coupled C0SP deep filtration membrane and Capto Adhere ImpRes chromatography column were equilibrated with more than 50L of 50mM Tris-HAc, pH 7.0, conductivity 10mS/cm, 100mM NaCl, and the loading operation was performed after completion of the equilibration.
(3) The sample is a monoclonal antibody, and the conditions of the sample loading are that the pH is 7.0 and the conductivity is 10 mS/cm; loading a sample to a deep filtration membrane at a flow rate of 200LMH by a peristaltic pump, and directly loading the sample to a Capto Adhere ImpRes chromatographic column after the sample is subjected to deep filtration by a C0SP deep filtration membrane; monitoring the pressure before the deep filtration membrane and the mixed-mode chromatographic column in the sample loading process, keeping the pressure within the range of 0-2 Bar, and when the pressure rises beyond the range, properly reducing the flow rate to about 50LMH to restore the pressure to the range;
the sample flow-through was collected at the beginning of the loading, i.e. at the outlet end of the Capto Adhere ImpRes chromatography column, and the loading was terminated after the loading reached 300 g/L.
(4) After the loading was completed, the C0SP deep filtration membrane and the sample remaining inside the Capto Adhere ImpRes chromatography column were top washed out by a peristaltic pump using more than 50L of the equilibration buffer, and finally the flow-through sample treated with the deep filtration treatment and mixed mode chromatography column was harvested.
(5) After the collection is completed, the connection between the deep filtration membrane and the mixed-mode chromatographic column is disconnected, the deep filtration membrane is discarded, then the used mixed-mode chromatographic column is regenerated by using 1M HAc aqueous solution, the regenerated mixed-mode chromatographic column is disinfected by using 0.5M NaOH aqueous solution, and then the regenerated and disinfected mixed-mode chromatographic column is preserved by using preservation solution for subsequent purification.
The results are shown in Table 2. The control group adopts a common purification process, the deep filtration and mixed mode chromatography are carried out step by step, and the experimental group adopts a continuous flow process. By comparison with the conventional process, the productivity of the present embodiment is improved by 47%, and the product quality is kept consistent.
Table 2 example 2 experimental results
Example 3
The protein purification system of this example, which integrates depth filtration with an anion exchange chromatography membrane in flow-through mode and a cation exchange chromatography column in flow-through mode, comprises a depth filtration membrane, an anion exchange chromatography membrane, a cation exchange chromatography column and a peristaltic pump; the outlet of the peristaltic pipe of the peristaltic pump is connected to the inlet of the deep filtration membrane, the outlet of the deep filtration membrane is connected to the inlet of the anion exchange chromatography membrane, and the outlet of the anion exchange chromatography membrane is connected to the inlet of the cation exchange chromatography column. Pressure gauges are arranged between the peristaltic pump and the deep filtration membrane, between the deep filtration membrane and the anion exchange chromatographic membrane, and between the anion exchange chromatographic membrane and the cation exchange chromatographic column.
Specifically, the deep filtration membrane is an X0HC deep filtration membrane of Merck, and the anion exchange chromatography membrane is an anion exchange chromatography membrane of Merck
HD-Q anion exchange chromatography membrane, the cation exchangeThe chromatographic column is
CP-FT column packed with Merck's packing
CP-FT。
This example provides a method of protein purification comprising the steps of:
(1) using 1.1m2X0HC deep filtration Membrane, 115mL Natrix HD-Q anion exchange chromatography Membrane and 1L
Washing the deep filtration membrane with 110L pure water by using a CP-FT chromatographic column (the column height is 20 cm); subjecting a Natrix HD-Q anion exchange chromatography membrane and
CP-FT chromatography column connected, and Natrix HD-Q anion exchange chromatography membrane treated with 0.5M NaOH aqueous solution and
and (3) simultaneously carrying out alkaline washing sterilization on the CP-FT chromatographic column, and connecting the outlet of the X0HC deep filtration membrane with the inlet of a Natrix HD-Q anion exchange chromatographic membrane after washing and sterilization are finished.
(2) Equilibrating the well-coupled X0HC deep filtration membrane, Natrix HD-Q anion exchange chromatography membrane and membrane using more than 60L of 50mM NaAc-HAc solution at pH 5.5
And (4) carrying out a CP-FT chromatographic column, and carrying out sample loading operation after the balance is finished.
(3) The sample is Fc fusion protein, and the conditions of the sample loading are that the pH is 5.5 and the conductivity is less than 5 mS/cm; loading the sample to a deep filtration membrane by a peristaltic pump at a flow rate of 200LMH, and directly loading the sample to a Natrix HD-Q anion exchange chromatographic membrane and a reverse osmosis membrane after the sample is subjected to deep filtration by an X0HC deep filtration membrane
A CP-FT chromatographic column; monitoring the pressure before the deep filtration membrane, the anion exchange chromatographic membrane and the chromatographic column in the sample loading process, keeping the pressure within the range of 0-1 Bar, and properly reducing the flow rate to about 50LMH when the pressure rises to exceed the range so as to restore the pressure to the range;
at the beginning of the loading, i.e. at the time of said
Collecting sample flow-through liquid at the outlet end of the CP-FT chromatographic column, wherein the sample loading amount reaches 1000g/m2After that, the sample application is ended.
(4) After loading was complete, X0HC deep filtration membrane, Natrix HD-Q anion exchange chromatography membrane and
and (3) washing out the sample remained in the CP-FT chromatographic column, and finally harvesting the flow-through sample subjected to deep filtration treatment, anion exchange chromatography treatment and cation exchange chromatography treatment.
(5) After the collection is completed, the connection between the deep filtration membrane and the anion exchange chromatography membrane is disconnected, the deep filtration membrane is discarded, then the anion exchange chromatography membrane and the cation exchange chromatography column are simultaneously sterilized with an aqueous solution of 0.5M NaOH, and then the sterilized anion exchange chromatography membrane and the sterilized cation exchange chromatography column are stored with a sufficient amount of a preservation solution for subsequent purification. After the preservation, the connection between the anion exchange chromatography membrane and the cation exchange chromatography column is disconnected.
The results are shown in Table 3. The control group adopts a common purification process, the deep filtration and mixed mode chromatography are carried out step by step, and the experimental group adopts a continuous flow process. By comparison with the conventional process, the productivity of the present embodiment is improved by 56%, and the product quality is kept consistent.
Table 3 example 3 experimental results
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 (20)
1. A system for protein purification comprising a depth filtration unit, a chromatography unit, and a peristaltic pump;
the outlet of a peristaltic pump pipe of the peristaltic pump is connected with the inlet of the deep layer filtering unit; the outlet of the depth filtration unit is directly connected to the inlet of the chromatography unit;
the chromatography unit comprises a chromatographic membrane and/or a chromatographic column; the depth filtration unit comprises a depth filtration membrane;
the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatographic membrane is 1m2﹕（100~110）mL；
The ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2﹕（0.8~10）L；
When the chromatography unit is a chromatography column, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2H (8.5-9.5) L; when the chromatography unit comprises a chromatography column and a chromatography membrane, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1m2﹕（0.8~1）L；
The number of the peristaltic pumps is one.
2. The system for protein purification according to claim 1, wherein the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatography membrane is 1m2﹕（102~108）mL。
3. The system for protein purification according to claim 1, wherein the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatography membrane is 1m2﹕（104~105）mL。
4. The system for protein purification according to claim 1, wherein the ratio of the membrane area of the deep filtration membrane to the membrane volume of the chromatography membrane is 1.1m2﹕115mL。
5. The system for protein purification according to any one of claims 1 to 4, wherein when the chromatography unit is a chromatography column, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1.1m2﹕9.8L。
6. The system for protein purification according to any one of claims 1 to 4, wherein when the chromatography unit comprises a chromatography column and a chromatography membrane, the ratio of the membrane area of the deep filtration membrane to the column volume of the chromatography column is 1.1m2﹕1L。
7. The system for protein purification according to claim 1, wherein the chromatography unit comprises one chromatography membrane or a plurality of chromatography membranes connected in series;
alternatively, the chromatography unit comprises one chromatography column or a plurality of chromatography columns connected in series;
or, the chromatography unit comprises a chromatography membrane and a chromatography column which are connected in series;
or the chromatography unit comprises a chromatographic membrane and a plurality of chromatographic columns connected in series, wherein the chromatographic membrane and the plurality of chromatographic columns connected in series are connected in series;
or the chromatography unit comprises a plurality of chromatographic membranes and a chromatographic column which are connected in series, wherein the plurality of chromatographic membranes and the chromatographic column are connected in series.
8. The system for protein purification according to claim 1, further comprising at least one pressure gauge.
9. The system for protein purification according to claim 8, wherein the pressure gauge is disposed between the peristaltic pump and the depth filtration unit.
10. The system for protein purification according to claim 1, wherein the chromatography membrane comprises an anion exchange chromatography membrane; the chromatography column comprises any one or more of a mixed mode chromatography column, an anion exchange chromatography column and a cation exchange chromatography column.
11. The protein purification system according to claim 10, wherein the anion exchange chromatography membranes are Natrix HD-Q anion exchange chromatography membranes.
12. The protein purification system according to claim 10, wherein the chromatography columns are Capto Adhere ImpRes chromatography columns or Eshmuno CP-FT chromatography columns.
13. The system for protein purification according to claim 10, wherein the deep filtration membrane is any one of A1HC deep filtration membrane, C0SP deep filtration membrane and X0HC deep filtration membrane.
14. A method of protein purification using the system for protein purification according to any one of claims 1 to 13, comprising the steps of:
after a sample to be purified is loaded by a peristaltic pump, carrying out deep filtration treatment and chromatography treatment by the deep filtration unit and the chromatography unit respectively; the chromatography is flow-through mode chromatography.
15. The method for protein purification of the system for protein purification according to claim 14, wherein the flow-through mode chromatography is one-step flow-through mode chromatography or multi-step flow-through mode chromatography.
16. The method of protein purification of the system of protein purification according to claim 14, wherein the flow rate is adjusted such that the pressure between the depth filtration process and the chromatography process is 0-2 Bar before the depth filtration process.
17. The method of protein purification of the system of protein purification according to claim 16, wherein the flow rate is adjusted such that the pressure between the depth filtration process and the chromatography process is > 0Bar and ≦ 1Bar before the depth filtration process.
18. The method for protein purification according to claim 16, wherein the flow rate of the depth filtration treatment is 50 to 200LMH, and the flow rate of the chromatography treatment is 20 to 50mL/min or 200 to 300 cm/hr.
19. The method of protein purification of the system for protein purification according to claim 14, wherein the depth filtration unit is subjected to a water washing process prior to the depth filtration process; sterilizing the chromatography unit prior to the depth filtration process.
20. The method of protein purification of the system for protein purification according to claim 19, wherein the depth filtration unit subjected to the water washing process and the chromatography unit subjected to the sterilization process are equilibrated with an equilibration buffer before the depth filtration process.
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