CN111318077A - Multiplex convection chromatographic system and method for purifying protein by using same - Google Patents
Multiplex convection chromatographic system and method for purifying protein by using same Download PDFInfo
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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
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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Abstract
The invention discloses a multiplexing convection chromatography system and a method for purifying protein by using the same, belonging to the technical field of protein purification. The multiplex convection chromatographic system comprises a feeding liquid storage tank, a peristaltic pump I, a tangential flow filtering device, a circulating liquid storage tank, a peristaltic pump II, a peristaltic pump III, a waste filtrate storage tank, a peristaltic pump IV, a peristaltic pump V, a macroporous continuous chromatographic device and a central processing unit. The invention also provides a method for purifying protein by using the multiplex convection chromatographic system. The system integrates the concentration of cell supernatant, buffer solution replacement, purification and the formula of the final product, and realizes one-step operation of recovering the final product from cell sap. The system can further integrate ultraviolet light detection, pH, conductivity meters and the like to realize real-time monitoring of the whole purification process, and finally establish a laboratory and pilot scale antibody production platform which is efficient, simple, convenient and economical, and has great application prospect in various protein purification pilot scale platforms.
Description
Technical Field
The invention relates to a multiplexing convection chromatography system and a method for purifying protein by using the same, belonging to the technical field of protein purification.
Background
Tangential Flow Filtration (TFF) is a new type of concentration filtration technology, which refers to a filtration mode in which the liquid flow direction is perpendicular to the filtration direction. Compare in traditional dead end filtration (dead end), the shearing force is produced on the filter medium surface to liquid flow among the tangential flow filtration system, has reduced the pile up of filter cake layer or gel layer, has guaranteed stable filtration speed, can effectively improve the treatment effeciency. The separation method uses membranes to separate components in liquid solutions or suspensions based on size, molecular weight, or other differences. The tangential flow filtration technology has the advantages of simple use, rapidness, high efficiency, scale enlargement or reduction, synchronous concentration and washing filtration, economy, time saving and the like. Tangential flow filtration technology can be integrated with a closed circulation flow path to achieve an efficient, sterile and rapid process, and is therefore commonly used for the separation of biologically active substances, the production of biologicals, blood products and vaccines.
Affinity chromatography is a common protein purification technique, in which a medium capable of specifically binding with a bioactive substance (such as an antibody) is used as a stationary phase, so that the corresponding bioactive substance in a liquid phase is selectively bound to the stationary phase, thereby separating from other impurities in the liquid phase and achieving the purpose of separation and purification. Macroporous continuous chromatography (AMC for short) is a novel affinity chromatography technique, the stationary phase of which consists of a single continuous porous material. Common stationary phase materials include agarose (agarose), silica (silica), glycidyl methacrylate/ethylene glycol dimethacrylate (glyddyl methacrylate/ethylene dimethacrylate), and Cryogels, among others. Macroporous continuous chromatography has many advantages, such as can be made into various forms and packed in columns, capillaries and microfluidic devices; the back pressure is low, and the liquid phase can flow through at high speed, so that the aims of quick separation and analysis are fulfilled; the stationary phase can be modified according to the requirement.
Monoclonal antibodies are specific antibodies produced by lymphocytic hybridomas that are directed against only a single epitope on the composite antigen molecule. Monoclonal antibodies have the advantages of high specificity and high homogeneity, and have been rapidly applied to a variety of fields including testing medical diagnostic reagents, radioimmunoassay technology, protein purification, and treatment of diseases such as tumor targeting, autoimmune diseases, infectious diseases, transplant rejection and the like. The conventional monoclonal antibody purification system is an AKTA protein purification system, has the advantage of high automation degree, and can realize integration of automatic sample loading, purification, sample collection and sample analysis. However, this system has the disadvantage that only one column, such as Protein A column, anion/cation exchange column, hydrophobic interaction column, etc., can be connected for each purification. When the components of the monoclonal antibody cell fermentation broth are complex, 3-4 chromatographic columns are needed for purification, and when the AKTA protein purification system is used for purifying the monoclonal antibody, each chromatographic column needs to be replaced by another chromatographic column after the use of the chromatographic column is finished, and the sample conditions such as conductivity, pH and the like need to be adjusted firstly when a new chromatographic column is used each time. Therefore, the existing monoclonal antibody preparation technology is complex, time-consuming and labor-consuming, and the price of the monoclonal antibody is also high.
Disclosure of Invention
In order to solve the problems that an AKTA protein purification system can only be connected with one chromatographic column each time, when the protein with complex components is purified, each chromatographic column needs to be replaced with another chromatographic column after use, and the sample condition needs to be readjusted, so that the process is complex, time-consuming and labor-consuming, the invention provides a multiplex convective chromatography system (MCC for short) and a method for purifying the protein by the same. The technical scheme is as follows:
the invention aims to provide a multiplexing convection chromatographic system which comprises a feeding liquid storage tank 1, a peristaltic pump I2, a tangential flow filtering device 3, a circulating liquid storage tank 4, a peristaltic pump II5, a peristaltic pump III6, a waste filtrate storage tank 7, a peristaltic pump IV8, a macroporous continuous chromatographic device 9, a peristaltic pump V13 and a central processing unit; wherein: the feeding liquid storage tank 1 is connected with a feeding hole of the tangential flow filtering device 3 through a peristaltic pump I2; the tangential flow filtration device 3 is provided with two discharge ports, namely a recovered filtrate discharge port and a waste filtrate discharge port; the waste filtrate discharge port is connected with a waste filtrate liquid storage tank 7 through a peristaltic pump III 6; the recovered filtrate discharge port is divided into two branches, one branch is connected with the feed port of the circulating liquid storage tank 4 through a peristaltic pump V13, and the other branch is connected with the total feed port of the macroporous continuous chromatography device 9 through a peristaltic pump IV 8; the discharge hole of the circulating liquid storage tank 4 is connected with the feed inlet of the tangential flow filtering device 3 through a peristaltic pump II 5; the total discharge hole of the macroporous continuous chromatographic device 9 is connected with the feed inlet of the circulating liquid storage tank 4; the central processor controls the starting and stopping of the peristaltic pump I2, the peristaltic pump II5, the peristaltic pump III6, the peristaltic pump IV8 and the peristaltic pump V13.
Preferably, the multiplexed convection chromatography system is a single channel multiplexed convection chromatography system, wherein the macroporous continuous chromatography device 9 comprises a macroporous continuous chromatography column.
More preferably, the macroporous continuous chromatography column is selected from any one of a hydrophobic chromatography column, an affinity chromatography column, a molecular sieve, an anion exchange chromatography column, a cation exchange chromatography column and a composite chromatography column.
Preferably, the multiplexed convective chromatography system is a multichannel multiplexed convective chromatography system, wherein the macroporous continuous chromatography device 9 comprises a plurality of macroporous continuous chromatography columns connected in parallel.
More preferably, the plurality of macroporous continuous chromatography columns connected in parallel are selected from any one of a hydrophobic chromatography column, an affinity chromatography column, a molecular sieve, an anion exchange chromatography column, a cation exchange chromatography column and a composite chromatography column connected in parallel or any plurality of types connected in parallel.
Preferably, the feed stock tank 1 is used to store a crude protein solution to be purified, a buffer and a stock solution of proteins.
Preferably, a pressure sensor 10 is arranged on a pipeline between the peristaltic pump I2 and the feed inlet of the tangential flow filtration device 3, and/or a pressure sensor 10 is arranged on a pipeline between the peristaltic pump II5 and the feed inlet of the tangential flow filtration device 3, and/or a pressure sensor 10 is arranged between the peristaltic pump IV8 and the total feed inlet of the macroporous continuous chromatography device 9, and/or a pressure sensor 10 is arranged between the waste filtrate outlet of the tangential flow filtration device 3 and the peristaltic pump III6, all the pressure sensors 10 are in data connection with the central processing unit, and collected pressure signals are transmitted to the central processing unit.
Preferably, a pH and conductivity sensor 12 is arranged between the peristaltic pump I2 and the feeding liquid storage tank 1, and/or a pH and conductivity sensor 12 is arranged between the peristaltic pump II5 and the discharge hole of the circulating liquid storage tank 4; the pH and conductivity sensor 12 is in data connection with a central processor, and the pH and conductivity sensor 12 transmits signals acquired by the pH and conductivity sensor to the central processor.
Preferably, the multiplex convection chromatography system further comprises a weighing device 11, wherein the weighing device 11 is connected with the circulating liquid storage tank 4 and is used for weighing the circulating liquid storage tank 4 and/or the solution stored in the circulating liquid storage tank.
More preferably, the weighing device 11 is an electronic scale.
More preferably, the weighing device 11 is a weight sensor, and the weight sensor acquires weight information of the circulating liquid storage tank 4 and the solution stored therein, and transmits a weight signal acquired by the weight sensor to the central processing unit.
The invention also provides a method for purifying protein by using the multiplex convection chromatographic system, which comprises the following steps:
1) conveying the crude protein solution to be purified to a tangential flow filtering device 3 for filtering, conveying waste filtrate to a waste filtrate liquid storage tank 7 by a peristaltic pump III6, conveying recovered filtrate to a circulating liquid storage tank 4, weighing the recovered filtrate in the circulating liquid storage tank 4, conveying the weighed recovered filtrate to the tangential flow filtering device 3 by a peristaltic pump II5, repeating the filtering process until a set concentration degree is reached, and finishing the concentration in the first stage;
2) replacing the solution in the feeding liquid storage tank 1 with buffer solution, conveying the buffer solution in the feeding liquid storage tank 1 to the tangential flow filtering device 3 by a peristaltic pump I2 for buffer solution replacement, conveying the replaced waste liquid to a waste filtrate liquid storage tank 7 by a peristaltic pump III6 in the replacement process, conveying the replaced recovery liquid to a circulating liquid storage tank 4, weighing the recovered recovery liquid, conveying the weighed recovered recovery liquid to the tangential flow filtering device 3 by a peristaltic pump II5, and repeating the replacement process to finish buffer solution replacement to a set pH value and conductivity;
3) conveying the solution in the tangential flow filtering device 3 and the circulating liquid storage tank 4 to a macroporous continuous chromatography device 9 for impurity removal;
4) replacing the solution in the feeding liquid storage tank 1 with a storage solution of protein, and conveying the solution subjected to impurity removal by the macroporous continuous chromatography device 9 into the tangential flow filtration device 3 again for repeated circulation to complete secondary concentration and replacement of the storage solution of protein;
5) and after the secondary concentration is finished, collecting the solution to obtain the purified protein solution.
More preferably, the method comprises the steps of:
1) storing a crude protein solution to be purified in a feeding liquid storage tank 1, starting a peristaltic pump I2, a peristaltic pump II5, a peristaltic pump III6 and a peristaltic pump V13 through a central processing unit, closing a peristaltic pump IV8, conveying the crude protein solution to be purified in the feeding liquid storage tank 1 to a tangential flow filtering device 3 for filtering, conveying waste filtrate to a waste filtrate liquid storage tank 7 through a peristaltic pump III6 in the filtering process, conveying recovered filtrate to a circulating liquid storage tank 4, weighing the recovered filtrate, conveying the weighed filtrate back to the tangential flow filtering device 3 through a peristaltic pump II5, repeating the filtering process until a set concentration degree is reached, and finishing the concentration in the first stage;
2) replacing the solution in the feeding liquid storage tank 1 with buffer solution, conveying the buffer solution in the feeding liquid storage tank 1 to the tangential flow filtering device 3 by a peristaltic pump I2 for buffer solution replacement, conveying the replaced waste liquid to a waste filtrate liquid storage tank 7 by a peristaltic pump III6 in the replacement process, conveying the replaced recovery liquid to a circulating liquid storage tank 4, weighing the recovered recovery liquid, conveying the weighed recovered recovery liquid to the tangential flow filtering device 3 by a peristaltic pump II5, and repeating the replacement process to finish buffer solution replacement to a set pH value and conductivity;
3) starting the peristaltic pump II5 and the peristaltic pump IV8 through the central processing unit, closing the peristaltic pump V13, enabling the solution in the tangential flow filtering device 3 and the circulating liquid storage tank 4 to enter the macroporous continuous chromatography device 9 for impurity removal, and enabling the solution after impurity removal through the macroporous continuous chromatography device 9 to enter the circulating liquid storage tank 4 for storage;
4) starting a peristaltic pump I2, a peristaltic pump II5, a peristaltic pump III6 and a peristaltic pump V13 through a central processing unit, closing a peristaltic pump IV8, replacing the solution in a feeding liquid storage tank 1 with a protein storage liquid, conveying the solution which is stored in a circulating liquid storage tank 4 and subjected to impurity removal through a macroporous continuous chromatography device 9 into a tangential flow filtration device 3 for repeated circulating treatment to complete secondary concentration and replacement of the protein storage liquid, and storing the finally treated liquid by the circulating liquid storage tank 4;
5) and (3) closing the peristaltic pump I2, the peristaltic pump II5, the peristaltic pump III6, the peristaltic pump V13 and the peristaltic pump IV8 through the central processor, and collecting the solution in the circulating liquid storage tank 4 to obtain the purified protein solution.
Preferably, the crude protein solution to be purified is a monoclonal antibody cell fermentation broth. More preferably, the concentration of the secondary concentration in the step 5) is 1-5 mg/mL.
The multiplex convection chromatographic system comprises a tangential flow filtration system (TFF) and a macroporous continuous chromatography (AMC), a single-channel multiplex convection chromatographic system and a multi-channel multiplex convection chromatographic system can be developed according to the complexity of the components of the monoclonal antibody cell fermentation liquor, the components of the monoclonal antibody cell fermentation liquor are simple, and the single-channel multiplex convection chromatographic system can be used for realizing one-step operation from cell liquor recovery to final product purification; under the condition of complex components of monoclonal antibody cell fermentation liquor, a multichannel multiplexing convection chromatography system can be used for realizing one-step operation from cell liquor recovery to final product purification.
The single-channel multiplexing convection chromatography system can form a circulation flow path by a tangential flow filtration device, a macroporous continuous chromatographic column, five peristaltic pumps, three pressure sensors, two pH/conductivity sensors, a circulation liquid storage tank, a feeding liquid storage tank and a waste filtrate storage tank. A weighing device and a central processing unit are externally connected to control the flow of the circulating liquid and the opening and closing of the peristaltic pump.
The multichannel multiplexing convection chromatographic system can form a circulating flow path by the components of a tangential flow filtration device, a plurality of macroporous continuous chromatographic columns, five peristaltic pumps, three pressure sensors, two pH/conductivity sensors, a circulating liquid storage tank, a feeding liquid storage tank and a waste filtrate storage tank outlet. A weighing device and a central processing unit are externally connected to control the flow of the circulating liquid and the opening and closing of the peristaltic pump.
The multiplex convection chromatographic system can be used for purifying proteins, is particularly suitable for monoclonal antibodies, and the method for processing the monoclonal antibodies by utilizing the multiplex convection chromatographic system comprises the following steps: feeding the monoclonal antibody cell fermentation liquor into a multiplex convection chromatographic system through a feed inlet, and firstly, feeding the monoclonal antibody cell fermentation liquor into a tangential flow filtration system for sample concentration; when the sample is concentrated to a certain degree, adding a buffer solution from the feed port to perform buffer solution replacement; the sample after being concentrated by a tangential flow filtration system and replaced by buffer liquid enters a macroporous continuous chromatography for one-step or multi-step impurity removal; after macroporous continuous chromatography treatment, the sample enters the tangential flow filtration system again for concentration. In the secondary concentration process, adding a monoclonal antibody storage solution from a feed inlet, and performing storage solution replacement on the sample; after the sample is displaced by the storage liquid and concentrated to a specific concentration range, the tangential flow filtration system and the macroporous continuous chromatography system are off-line, and the sample is collected and stored by the collecting pipe.
The buffer composition in the invention is determined according to the adsorption medium of macroporous continuous chromatography.
The crude protein solution to be purified in the invention is a crude protein solution obtained by primarily purifying the obtained fermentation cells after centrifugation, cell disruption, centrifugation and filtration.
The storage solution of the protein in the invention refers to a solution for storing the purified protein.
The macroporous continuous chromatographic column in the invention refers to a protein chromatographic column based on macroporous continuous chromatographic technology, namely the chromatographic column is a purification column combining macroporous continuous chromatographic technology and protein chromatography, such as a hydrophobic chromatographic column, an affinity chromatographic column, a molecular sieve, an anion exchange chromatographic column, a cation exchange chromatographic column, a composite chromatographic column or other protein chromatographic columns based on macroporous continuous chromatographic technology. The invention has the beneficial effects that:
the tangential flow filtration device has the functions of concentration, buffer solution replacement and protein storage solution replacement, and the macroporous continuous chromatography device has the purification function, so that the system fully utilizes the high flux and the strong pressure resistance of the chromatographic column, combines the tangential flow filtration system, and integrates the concentration of cell supernatant, the buffer solution replacement, the purification and the formula of a final product; meanwhile, the system integrates the downstream purification steps, does not need to replace a chromatographic column, readjusts conditions, realizes one-step operation of recovering the final product from the cell sap, and can achieve the purposes of time saving, labor saving, economy and high efficiency in purification. In addition, the system can further integrate ultraviolet light detection, pH, conductivity meters and the like to realize real-time monitoring of the whole purification process, and finally establish a laboratory and pilot scale antibody production platform which is efficient, simple, convenient and economical, and has great application prospect in various protein purification pilot scale platforms.
The system of the invention has better purification effect, such as processing monoclonal antibody protein, and the content of Host Cell Protein (HCP) is 300,000ppm and the content of DNA is 30,000ppm in cell fermentation broth before purification. After pretreatment of caprylic acid-allantoin-centrifugation-filtration, the content of HCP is reduced to be below 500ppm, and after purification by a single-channel multiplexing convection chromatography system, the content of HCP is less than 100ppm, and the content of DNA is less than 10 ppm; the purity of the IgG1 is more than 95%, and the recovery rate is higher than 85%.
Drawings
FIG. 1 is a schematic diagram of a single-channel multiplexed convection chromatograph;
FIG. 2 is a schematic diagram of purification of single channel multiplexed convection chromatography;
FIG. 3 is a schematic diagram of a multichannel multiplexed convection chromatograph configuration;
FIG. 4 is a schematic diagram of purification of multi-channel multiplexed convection chromatography;
in the figure: 1, a feeding liquid storage tank; 2, a peristaltic pump I; 3, a tangential flow filtration device; 4, a circulating liquid storage tank; 5, a peristaltic pump II; 6, peristaltic pump III; 7, a waste filtrate storage tank; 8, a peristaltic pump IV; 9, a macroporous continuous chromatography device; 10, a pressure sensor; 11, a weighing device; 12, pH and conductivity sensors; 13, peristaltic pump V.
Detailed Description
The present invention will be further described with reference to the following specific examples, but the present invention is not limited to these examples.
Example 1
As shown in fig. 1, the embodiment provides a single-channel multiplexing convection chromatography system, which includes a feeding liquid storage tank 1, a peristaltic pump I2, a tangential flow filtration device 3, a circulating liquid storage tank 4, a peristaltic pump II5, a peristaltic pump III6, a waste filtrate storage tank 7, a peristaltic pump IV8, a macroporous continuous chromatography device 9, a peristaltic pump V13 and a central processing unit; wherein: the feeding liquid storage tank 1 is connected with a feeding hole of the tangential flow filtering device 3 through a peristaltic pump I2; the tangential flow filtration device 3 is provided with two discharge ports, namely a recovered filtrate discharge port and a waste filtrate discharge port; the waste filtrate discharge port is connected with a waste filtrate liquid storage tank 7 through a peristaltic pump III 6; the recovered filtrate discharge port is divided into two branches, one branch is connected with the feed port of the circulating liquid storage tank 4 through a peristaltic pump V13, and the other branch is connected with the total feed port of the macroporous continuous chromatography device 9 through a peristaltic pump IV 8; the discharge hole of the circulating liquid storage tank 4 is connected with the feed inlet of the tangential flow filtering device 3 through a peristaltic pump II 5; the total discharge hole of the macroporous continuous chromatographic device 9 is connected with the feed inlet of the circulating liquid storage tank 4; the central processor controls the starting and stopping of the peristaltic pump I2, the peristaltic pump II5, the peristaltic pump III6, the peristaltic pump IV8 and the peristaltic pump V13.
The multiplex convection chromatography system of the present embodiment is a single channel multiplex convection chromatography system, wherein the macroporous continuous chromatography device 9 comprises a macroporous continuous chromatography column. The macroporous continuous chromatographic column is selected from any one of a hydrophobic chromatographic column, an affinity chromatographic column, a molecular sieve, an anion exchange chromatographic column, a cation exchange chromatographic column and a composite chromatographic column. The tangential flow filtration device 3 comprises a tangential flow filtration column.
The feeding liquid storage tank 1 of this embodiment is used for storing a crude protein solution to be purified, a buffer solution, and a protein storage solution, respectively.
As a preferable mode of the embodiment, pressure sensors 10 are arranged on a pipeline between the peristaltic pump I2 and the feed inlet of the tangential flow filtration device 3, a pipeline between the peristaltic pump II5 and the feed inlet of the tangential flow filtration device 3, a pipeline between the peristaltic pump IV8 and the total feed inlet of the macroporous continuous chromatography device 9, and a pipeline between the waste filtrate outlet and the peristaltic pump III 6; all pressure sensors 10 are in data connection with the central processing unit and transmit the acquired pressure signals to the central processing unit.
As a preferable mode of the embodiment, pH and conductivity sensors 12 are respectively arranged between the peristaltic pump I2 and the feeding liquid storage tank 1 and between the peristaltic pump II5 and the discharge hole of the circulating liquid storage tank 4; the pH and conductivity sensors 12 are in data connection with the central processor, and the pH/conductivity sensors transmit the signals they acquire to the central processor.
As a preferable mode of this embodiment, the multiplex convection chromatography system further includes a weighing device 11, and the weighing device 11 is connected to the circulating liquid storage tank 4 and is configured to weigh the solution in the circulating liquid storage tank 4. The weighing device 11 can be an electronic scale and directly weighs the weight of the circulating liquid storage tank 4 and the weight of the solution stored in the circulating liquid storage tank; the weighing device 11 can also be a weight sensor which collects weight information of the circulating liquid storage tank 4 and the solution stored therein and transmits the collected weight signal to the central processing unit.
The technical schematic diagram of the single-channel multiplexing convection chromatography system of the embodiment is shown in fig. 2.
The present example also provides a method for purifying a protein using the above-described multiplexed convection chromatography system, comprising the steps of:
1) storing a crude protein solution to be purified, such as monoclonal antibody cell fermentation liquor, in a feeding liquid storage tank 1, starting a peristaltic pump I2, a peristaltic pump II5, a peristaltic pump III6 and a peristaltic pump V13 through a central processing unit, closing a peristaltic pump IV8, conveying the crude protein solution to be purified in the feeding liquid storage tank 1 to a tangential flow filtering device 3 for filtering, conveying waste filtrate to a waste filtrate liquid storage tank 7 through a peristaltic pump III6 in the filtering process, conveying recovered filtrate to a circulating liquid storage tank 4, weighing by a weighing device 11, conveying the recovered filtrate back to the tangential flow filtering device 3 through a peristaltic pump II5, repeating the filtering process until a set concentration degree is reached, and finishing the concentration in a first stage;
2) replacing the solution in the feeding liquid storage tank 1 with buffer solution, conveying the buffer solution in the feeding liquid storage tank 1 to the tangential flow filtration device 3 by a peristaltic pump I2 for buffer solution replacement, conveying the replaced waste liquid to a waste filtrate liquid storage tank 7 by a peristaltic pump III6 in the replacement process, conveying the replaced recovery liquid to a circulating liquid storage tank 4, weighing by a weighing device 11, conveying the waste liquid to the tangential flow filtration device 3 by a peristaltic pump II5, and repeating the replacement process to complete buffer solution replacement to a set pH value and conductivity;
3) starting a peristaltic pump II5 and a peristaltic pump IV8 through a central processing unit, closing a peristaltic pump V13, enabling the solution in the tangential flow filtering device 3 and the circulating liquid storage tank 4 to enter a macroporous continuous chromatographic device 9 for impurity removal and purification, when the adsorption of pollutants approaches to balance, taking the macroporous continuous chromatographic device off the line, and enabling the solution after impurity removal through the macroporous continuous chromatographic device 9 to enter the circulating liquid storage tank 4 for storage;
4) starting a peristaltic pump I2, a peristaltic pump II5, a peristaltic pump III6 and a peristaltic pump V13 through a central processing unit, closing a peristaltic pump IV8, replacing the solution in a feeding liquid storage tank 1 with a protein storage liquid, conveying the solution which is stored in a circulating liquid storage tank 4 and subjected to impurity removal through a macroporous continuous chromatography device 9 into a tangential flow filtration device 3 for repeated circulating treatment to complete secondary concentration and replacement of the protein storage liquid, and storing the finally treated liquid by the circulating liquid storage tank 4;
5) when the concentration of the monoclonal antibody reaches 1mg/mL, the peristaltic pump I2, the peristaltic pump II5, the peristaltic pump III6, the peristaltic pump V13 and the peristaltic pump IV8 are closed through the central processing unit, and the solution in the circulating liquid storage tank 4 is collected, so that the purified protein solution is obtained.
Example 2
As shown in fig. 3, the present embodiment is different from embodiment 1 in that: the multiplex convection chromatographic system is a multichannel multiplex convection chromatographic system, wherein the macroporous continuous chromatographic device 9 comprises 2 macroporous continuous chromatographic columns connected in parallel. Wherein 2 macroporous continuous chromatographic columns connected in parallel are respectively an anion exchange chromatographic column and a hydrophobic chromatographic column. Or any combination of a hydrophobic chromatography column, an affinity chromatography column, a molecular sieve, an anion exchange chromatography column, a cation exchange chromatography column and a composite chromatography column.
The technical schematic diagram of the multichannel multiplexing convection chromatography system of the embodiment is shown in FIG. 4.
The purification method of this example differs from example 1 in that: and when the concentration of the monoclonal antibody reaches 5mg/mL, taking the TFF system off the line, finishing the sample treatment, and finally collecting and storing the purified sample by a collecting pipe.
Example 3
The monoclonal antibody IgG1 is taken as an example to show that the single-channel multiplexing convection chromatography system can complete one-step operation from concentration, recovery and purification of cell fermentation liquor.
Cell culture: monoclonal antibody IgG1 was produced by expression from Chinese Hamster Ovary Cells (CHO), the antibody being present at 5 liters
B production in stirred glass bioreactor by fed-batch processAnd (3) a culture mode, wherein cell fermentation liquor is collected after 15-30 days of culture. The cell culture medium is protein-free medium CD CHO (Life technologies) and protein-free medium
PF-CHO (GE healthcare) in a volume ratio of 1: 1.
Pre-treating cell fermentation liquid, adjusting pH of the cell fermentation liquid to 5.3, adding caprylic acid and allantoin to make final mass concentration of caprylic acid and allantoin in the mixture 0.4% and 1%, respectively, stirring the mixture at room temperature for 2 hr, centrifuging at 4000 × g at room temperature for 20min, collecting supernatant, and filtering with 0.22 μm filter membrane (filter membrane: (filter membrane)
Rapid-Flow Filters, Thermo Scientific) and the filtrate was collected.
The filtrate (about 5 liters) is placed in a feeding liquid storage tank 1, enters a tangential flow filtration device 3 (a tangential flow filtration column, TFF) through a peristaltic pump I, the waste filtrate is discharged out of the system through a peristaltic pump III6 waste filtrate storage tank 7, and the sample concentrated by the tangential flow filtration device 3 enters a circulating liquid storage tank 4, is weighed and then enters the TFF column again for concentration. When the volume of the circulating solution is concentrated to about 3 liters, Tris-HCl buffer (pH 7.5) is added to the feed stock tank 1 and enters the TFF column to perform buffer replacement on the concentrated sample. After the circulating liquid is replaced by the buffer solution, the central processor controls the peristaltic pump IV8 at the front end of the macroporous continuous chromatographic device 9 (a single-channel multiplexing convection chromatographic system, and an anion exchange chromatographic column is adopted as the macroporous continuous chromatographic column) to be started, the circulating liquid enters the macroporous continuous chromatographic device 9 (the anion exchange column) to start IgG1 purification, and after all the circulating liquid flows out of the macroporous continuous chromatographic device 9 (the anion exchange column), the AMC system is finished to be off-line after the purification. After purification IgG1 was further concentrated in the TFF system while the IgG1 was subjected to stock replacement by adding PBS buffer (pH 7.2-7.4,10mM) as a monoclonal antibody stock to feed stock tank 1. When the concentration of IgG1 reached 3mg/mL, the TFF system was taken off line, sample processing was terminated, and the final purified samples were collected by collection tubes and stored. The host protein content was determined by GenerDetecting with a kit of (ii) a CHO HCP (human serum albumin) and DNA content with a digital PCR (polymerase chain reaction) instrument QX100TMDroplet DigitalTMPCR System (Bio-Rad) assay.
The experimental results are as follows: the cell fermentation broth before purification had a Host protein (HCP) content of 300,000ppm and a DNA content of 30,000 ppm. After pretreatment of caprylic acid-allantoin-centrifugation-filtration, the HCP content was reduced to below 500 ppm. After purification by a single-channel multiplexing convection chromatography system, the content of HCP is less than 100ppm, and the content of DNA is less than 10 ppm; the purity of the IgG1 is more than 95%, and the recovery rate is higher than 85%. Meets the requirements of the existing Food and Drug Administration (FDA) on the protein product of the monoclonal antibody.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The multiplex convection chromatographic system is characterized by comprising a feeding liquid storage tank (1), a peristaltic pump I (2), a tangential flow filtering device (3), a circulating liquid storage tank (4), a peristaltic pump II (5), a peristaltic pump III (6), a waste filtrate storage tank (7), a peristaltic pump IV (8), a macroporous continuous chromatographic device (9), a peristaltic pump V (13) and a central processing unit; wherein: the feeding liquid storage tank (1) is connected with a feeding hole of the tangential flow filtration device (3) through a peristaltic pump I (2); the tangential flow filtering device (3) is provided with two discharge ports, namely a recovered filtrate discharge port and a waste filtrate discharge port; the waste filtrate discharge port is connected with a waste filtrate storage tank (7) through a peristaltic pump III (6); the recovered filtrate discharge port is divided into two branches, one branch is connected with the feed port of the circulating liquid storage tank (4) through a peristaltic pump V (13), and the other branch is connected with the total feed port of the macroporous continuous chromatography device (9) through a peristaltic pump IV (8); the discharge hole of the circulating liquid storage tank (4) is connected with the feed hole of the tangential flow filtering device (3) through a peristaltic pump II (5); the total discharge hole of the macroporous continuous chromatographic device (9) is connected with the feed inlet of the circulating liquid storage tank (4); the central processing unit controls the starting and stopping of the peristaltic pump I (2), the peristaltic pump II (5), the peristaltic pump III (6), the peristaltic pump IV (8) and the peristaltic pump V (13).
2. The multiplexed convective chromatography system of claim 1, wherein the multiplexed convective chromatography system is a single channel multiplexed convective chromatography system, wherein the macroporous continuous chromatography device (9) comprises one macroporous continuous chromatography column.
3. The multiplexed convective chromatography system of claim 2, wherein the macroporous continuous chromatography column is selected from any one of a hydrophobic chromatography column, an affinity chromatography column, a molecular sieve, an anion exchange chromatography column, a cation exchange chromatography column and a composite chromatography column.
4. The multiplexed convective chromatography system of claim 1, wherein the multiplexed convective chromatography system is a multichannel multiplexed convective chromatography system in which the macroporous continuous chromatography device (9) comprises a plurality of macroporous continuous chromatography columns connected in parallel.
5. The multiplexed convection chromatography system of claim 4, wherein the plurality of parallel-connected macroporous continuous chromatography columns are selected from any one or any plurality of parallel-connected hydrophobic chromatography columns, affinity chromatography columns, molecular sieves, anion exchange chromatography columns, cation exchange chromatography columns, and composite chromatography columns.
6. The multiplexed convection chromatography system of claim 1, wherein the feed reservoir (1) is configured to store a crude protein solution to be purified, a buffer, and a protein reservoir.
7. The system of claim 1, wherein a pressure sensor (10) is disposed in a pipeline between the peristaltic pump I (2) and the feed inlet of the tangential flow filtration device (3), and/or a pressure sensor (10) is disposed in a pipeline between the peristaltic pump II (5) and the feed inlet of the tangential flow filtration device (3), and/or a pressure sensor (10) is disposed between the peristaltic pump IV (8) and the total feed inlet of the macroporous continuous chromatography device (9), and/or a pressure sensor (10) is disposed between the waste filtrate outlet of the tangential flow filtration device (3) and the peristaltic pump III (6), and all the pressure sensors (10) are in data communication with the central processing unit to transmit the collected pressure signals to the central processing unit.
8. The multiplexed convection chromatography system of claim 1, wherein pH and conductivity sensors (12) are provided between the peristaltic pump I (2) and the feed liquid reservoir (1) and/or pH and conductivity sensors (12) are provided between the peristaltic pump II (5) and the discharge port of the recycle liquid reservoir (4); the pH and conductivity sensor (12) is in data connection with the central processing unit, and the pH and conductivity sensor (12) transmits signals acquired by the pH and conductivity sensor to the central processing unit.
9. A method for purifying a protein using the multiplexed convective chromatography system of claim 1, comprising the steps of:
1) conveying a crude protein solution to be purified to a tangential flow filtering device (3) for filtering, conveying waste filtrate to a waste filtrate liquid storage tank (7) by a peristaltic pump III (6), enabling recovered filtrate to flow to a circulating liquid storage tank (4), conveying the recovered filtrate in the circulating liquid storage tank (4) to the tangential flow filtering device (3) by a peristaltic pump II (5) after weighing, repeating the filtering process until a set concentration degree is reached, and finishing the concentration in the first stage;
2) replacing the solution in the feeding liquid storage tank (1) with a buffer solution, conveying the buffer solution in the feeding liquid storage tank (1) to a tangential flow filtering device (3) by a peristaltic pump I (2) for buffer solution replacement, conveying the replaced waste liquid to a waste filtrate liquid storage tank (7) by a peristaltic pump III (6) in the replacement process, conveying the replaced recovery liquid to a circulating liquid storage tank (4), weighing the recovered recovery liquid, conveying the weighed recovery liquid to the tangential flow filtering device (3) by a peristaltic pump II (5), and repeating the replacement process to finish buffer solution replacement to a set pH value and conductivity;
3) conveying the solution in the tangential flow filtering device (3) and the circulating liquid storage tank (4) to a macroporous continuous chromatography device (9) for impurity removal;
4) replacing the solution in the feeding liquid storage tank (1) with a protein storage solution, and conveying the solution subjected to impurity removal through the macroporous continuous chromatography device (9) to the tangential flow filtration device (3) again for repeated circulation treatment to complete secondary concentration and replacement of the protein storage solution;
5) and after the secondary concentration is finished, collecting the solution to obtain the purified protein solution.
10. The method according to claim 9, wherein the crude protein solution to be purified is a monoclonal antibody cell fermentation broth.
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| CN115282779A (en) * | 2022-07-27 | 2022-11-04 | 山东泰邦生物制品有限公司 | Unidirectional flow ultrafiltration system for realizing dialysis function and operation method thereof |
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