CN111318077A - A multiplexed convection chromatography system and method for purifying protein - Google Patents

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

A multiplexed convection chromatography system and method for purifying protein

technical field

The invention relates to a multiplexed convection chromatography system and a method for purifying protein thereof, belonging to the technical field of protein purification.

Background technique

Tangential flow filtration (TFF) is a new type of concentrated filtration technology, which refers to a filtration form in which the liquid flow direction is perpendicular to the filtration direction. Compared with the traditional dead end filtration, the liquid flow in the tangential flow filtration system generates shear force on the surface of the filter medium, which reduces the accumulation of the filter cake layer or gel layer and ensures a stable filtration rate. It can effectively improve the processing efficiency. This separation method uses membranes to separate components in a liquid solution or suspension based on size, molecular weight, or other differences. The tangential flow filtration technology has the advantages of simple use, rapidity, high efficiency, scale can be enlarged or reduced, simultaneous concentration and diafiltration, economical and time-saving. Tangential flow filtration technology can integrate a closed circulation flow path to obtain an efficient, sterile and fast process. Therefore, tangential flow filtration technology is often used for the separation of biologically active substances, and the production of biological products, blood products and vaccines.

Affinity chromatography is a common protein purification technology that uses a medium that can specifically bind to biologically active substances (such as antibodies) as a stationary phase, so that the corresponding biologically active substances in the liquid phase can be selectively bound to the stationary phase, thereby It is separated from other impurities in the liquid phase to achieve the purpose of separation and purification. Macroporous continuous chromatography (affinity monolithchromatography, referred to as AMC) is a new type of affinity chromatography technology, and its stationary phase is composed of a single continuous porous material. Common stationary phase materials include agarose, silica, glycidyl methacrylate/ethylene dimethacrylate (glycidyl methacrylate/ethylene dimethacrylate), and Cryogels. Macroporous continuous chromatography has many advantages, for example, it can be made into various forms to be filled in columns, capillaries and microfluidic devices; the back pressure is low, and the liquid phase can flow at high speed to achieve the purpose of rapid separation and analysis; stationary phase can be carried out according to needs. modification modification.

Monoclonal antibodies are specific antibodies that are produced by lymphocyte hybridomas and are only directed against a single antigenic determinant on the complex antigen molecule. Monoclonal antibodies have the advantages of high specificity and high homogeneity, and have been rapidly used in many fields, including laboratory medicine diagnostic reagents, radioimmunoimaging technology, protein purification and tumor targeting, autoimmune diseases, infectious diseases and transplantation Rejection and other diseases treatment. The commonly used monoclonal antibody purification system is the AKTA protein purification system, which has the advantage of high automation and can realize automatic sample loading, purification, sample collection, and sample analysis. However, the disadvantage of this system is that only one column can be connected for each purification, such as Protein A column, anion/cation exchange column, hydrophobic interaction column, etc. When the composition of the monoclonal antibody cell fermentation broth is complex, 3-4 chromatographic columns need to be used for purification. Using the AKTA protein purification system to purify monoclonal antibodies requires that each chromatographic column should be replaced with another chromatographic column after use. , and every time a new column is used, the sample conditions, such as conductivity, pH, etc., need to be adjusted first. Therefore, the current monoclonal antibody preparation technology is complicated, time-consuming and labor-intensive, resulting in a relatively high price of the monoclonal antibody.

SUMMARY OF THE INVENTION

In order to solve the problem that the AKTA protein purification system can only be connected to one chromatographic column for each purification. When purifying proteins with complex components, each chromatographic column needs to be replaced with another chromatographic column after use, and the sample conditions must be readjusted. The process of this method is complicated, time-consuming and labor-intensive. The present invention provides a multiplexed convective chromatography (MCC for short) and a method for protein purification. It is a one-step operation from liquid recovery to final product purification, which can be applied to laboratory and pilot-scale antibody production platforms. The technical solutions adopted are as follows:

The object of the present invention is to provide a multiplexing convection chromatography system, the multiplexing convection chromatography system comprises a feed liquid storage tank 1, a peristaltic pump I2, a tangential flow filter device 3, a circulating liquid storage tank 4, a peristaltic pump II5, Peristaltic pump III6, waste filtrate storage tank 7, peristaltic pump IV8, macroporous continuous chromatography device 9, peristaltic pump V13 and central processing unit; Wherein: described feed storage tank 1 is by peristaltic pump I2 and tangential flow filter device The feed ports of 3 are connected; the tangential flow filter device 3 is provided with two discharge ports, which are respectively a recovery filtrate discharge port and a waste filtrate discharge port; The filtrate storage tank 7 is connected; the reclaimed filtrate discharge port is divided into two branches, one branch is connected with the feed port of the circulating liquid storage tank 4 by the peristaltic pump V13, and the other branch is connected with the large by the peristaltic pump IV8. The total feed port of the continuous chromatographic device 9 is connected; the discharge port of the circulating liquid storage tank 4 is connected to the feed port of the tangential flow filter device 3 through the peristaltic pump II5; The total discharge port is connected with the feed port of the circulating liquid storage tank 4; the central processing unit controls the start and stop 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 includes a macroporous continuous chromatography column.

More preferably, the macroporous continuous chromatography column is selected from any one of hydrophobic chromatography column, affinity chromatography column, molecular sieve, anion exchange chromatography column, cation exchange chromatography column and composite chromatography column.

Preferably, the multiplexed convection chromatography system is a multi-channel multiplexed convection chromatography system, wherein the macroporous continuous chromatography device 9 includes 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 of hydrophobic chromatography columns, affinity chromatography columns, molecular sieves, anion exchange chromatography columns, cation exchange chromatography columns and composite chromatography columns. A parallel connection or any number of parallel connections.

Preferably, the feed storage tank 1 is used to store the crude protein solution to be purified, the buffer solution and the protein storage solution.

Preferably, a pressure sensor 10 is provided on the pipeline between the peristaltic pump I2 and the feed port of the tangential flow filter device 3, and/or on the pipeline between the peristaltic pump II5 and the feed port of the tangential flow filter device 3 A pressure sensor 10 is provided, and/or a pressure sensor 10 is provided between the peristaltic pump IV8 and the total feed port of the macroporous continuous chromatography device 9, and/or the waste filtrate outlet of the tangential flow filter device 3 and Pressure sensors 10 are arranged between the peristaltic pumps III6, all the pressure sensors 10 are connected to the central processing unit for data, and transmit the collected pressure signals to the central processing unit.

Preferably, a pH and conductivity sensor 12 is provided between the peristaltic pump I2 and the feed storage tank 1, and/or a pH and conductivity sensor 12 is provided between the peristaltic pump II5 and the discharge port of the circulating liquid storage tank 4 Rate sensor 12; the pH and conductivity sensor 12 is data-connected to the central processing unit, and the pH and conductivity sensor 12 transmits the collected signals to the central processing unit.

Preferably, the multiplexed convection chromatography system further includes a weighing device 11, which is connected to the circulating liquid storage tank 4 for weighing the circulating liquid storage tank 4 and/or the solution stored therein the weight of.

More preferably, the weighing device 11 is an electronic scale.

More preferably, the weighing device 11 is a weight sensor, which collects the 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 present invention also provides a method for purifying protein by utilizing the above-mentioned multiplexing convection chromatography system, the method comprising the following steps:

1) the crude protein solution to be purified is transported to the tangential flow filtration device 3 and filtered, the waste filtrate is transported to the waste filtrate storage tank 7 by the peristaltic pump III6, and the reclaimed filtrate flows into the circulating liquid storage tank 4, The reclaimed filtrate in the circulating liquid storage tank 4 is transported back to the tangential flow filtration device 3 by the peristaltic pump II5 after weighing, and the above-mentioned filtration process is repeated to reach a predetermined concentration degree, and the concentration of the first stage is completed;

2) the solution in the feed storage tank 1 is replaced with a buffer, the buffer in the feed storage tank 1 is transported to the tangential flow filter device 3 by the peristaltic pump 12 to carry out buffer replacement, in the replacement process The waste liquid after the replacement is transported to the waste filtrate storage tank 7 by the peristaltic pump III6, the recovered liquid after the replacement is transported to the circulating liquid storage tank 4, and is transported back to the tangential flow filtration by the peristaltic pump II5 after weighing. In device 3, the above-mentioned replacement process is repeated to complete the buffer replacement to a predetermined pH and conductivity;

3) the solution in the tangential flow filter device 3 and the circulating liquid storage tank 4 is transported to the macroporous continuous chromatography device 9 for impurity removal;

4) The solution in the feeding liquid storage tank 1 is replaced with the storage solution of the protein, and the solution after the impurity removal by the macroporous continuous chromatography device 9 is re-transported to the tangential flow filter device 3 for repeated circulation to complete the secondary concentration. and replacement of protein storage solutions;

5) After the secondary concentration is completed, the solution is collected to obtain the purified protein solution.

More preferably, the method includes the steps of:

1) the crude protein solution to be purified is stored in the feed storage tank 1, starts peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 by central processing unit, closes peristaltic pump IV8, and feeds storage. The crude protein solution to be purified in the liquid tank 1 is transported to the tangential flow filtration device 3 for filtration, and the waste filtrate is transported to the waste filtrate storage tank 7 by the peristaltic pump III6 during the filtration process, and the recovered filtrate is transported to the circulation In the liquid storage tank 4, after weighing, the peristaltic pump II5 is transported back to the tangential flow filter device 3, and the above-mentioned filtering process is repeated to reach a predetermined concentration level, and the concentration of the first stage is completed;

2) the solution in the feed storage tank 1 is replaced with a buffer, the buffer in the feed storage tank 1 is transported to the tangential flow filter device 3 by the peristaltic pump 12 to carry out buffer replacement, in the replacement process The waste liquid after the replacement is transported to the waste filtrate storage tank 7 by the peristaltic pump III6, the recovered liquid after the replacement is transported to the circulating liquid storage tank 4, and is transported back to the tangential flow filtration by the peristaltic pump II5 after weighing. In device 3, the above-mentioned replacement process is repeated to complete the buffer replacement to a predetermined pH and conductivity;

3) start peristaltic pump II5 and peristaltic pump IV8 by central processing unit, close peristaltic pump V13, make the solution in tangential flow filter device 3 and circulating liquid storage tank 4 enter macroporous continuous chromatographic device 9 and carry out impurity removal, after large The solution after the impurity removal of the hole continuous chromatography device 9 enters the circulating liquid storage tank 4 for storage;

4) start peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 by central processing unit, close peristaltic pump IV8, the solution in the feed storage tank 1 is replaced by the storage solution of protein, the circulating fluid storage solution The solution stored in the tank 4 after the impurity removal by the macroporous continuous chromatography device 9 is transported to the tangential flow filtration device 3 for repeated circulation treatment to complete the secondary concentration and replacement of the protein storage liquid, and the final treated liquid is replaced by the circulating liquid. storage tank 4;

5) close peristaltic pump I2, peristaltic pump II5, peristaltic pump III6, peristaltic pump V13 and peristaltic pump IV8 by central processing unit, collect the solution in the circulating fluid storage tank 4, promptly obtain the protein solution after purification.

Preferably, the crude protein solution to be purified is a monoclonal antibody cell fermentation broth. More preferably, the concentration of the concentrated solution in step 5) is 1-5 mg/mL by the secondary concentration.

The multiplexed convection chromatography system of the invention includes a tangential flow filtration system (TFF) and a macroporous continuous chromatography (AMC). According to the complexity of the components of the monoclonal antibody cell fermentation broth, a single-channel multiplexed convection chromatography system and a multi-channel complex With the convection chromatography system, when the composition of the monoclonal antibody cell fermentation broth is simple, the single-channel multiplexing convection chromatography system can realize one-step operation from the recovery of the cell liquid to the final product purification; when the composition of the monoclonal antibody cell fermentation broth is complex, use The multi-channel multiplexed convection chromatography system can realize one-step operation from cell fluid recovery to final product purification.

The single-channel multiplexed convection chromatography system can consist of a tangential flow filtration device, a macroporous continuous chromatography column, five peristaltic pumps, three pressure sensors, two pH/conductivity sensors, a circulating fluid reservoir, a A feed liquid storage tank and a waste filtrate liquid storage tank form a circulating flow path. An external weighing device and a central processing unit are connected to control the flow of circulating fluid and the opening and closing of the peristaltic pump.

The multi-channel multiplexed convection chromatography system can consist of a tangential flow filter device, multiple macroporous continuous chromatography columns, five peristaltic pumps, three pressure sensors, two pH/conductivity sensors, a circulating fluid reservoir, a The feed liquid storage tank and the outlet of a waste filtrate liquid storage tank form a circulating flow path. An external weighing device and a central processing unit are connected to control the flow of circulating fluid and the opening and closing of the peristaltic pump.

The multiplexed convection chromatography system of the present invention can be used for purifying proteins, and is especially suitable for monoclonal antibodies. The method for using the multiplexed convection chromatography system of the present invention to process the monoclonal antibodies is as follows: the monoclonal antibody cell fermentation broth is introduced into the complex through the feed port. With the convection chromatography system, first enter the tangential flow filtration system for sample concentration; when the sample is concentrated to a certain extent, add buffer from the feed port for buffer exchange; the sample after concentration and buffer exchange through the tangential flow filtration system Enter the macroporous continuous chromatography for one or more steps of impurity removal; after the macroporous continuous chromatography treatment, the sample re-enters the tangential flow filtration system for concentration. During the secondary concentration process, the monoclonal antibody storage solution was added from the feed port, and the sample was replaced by the storage solution; after the sample was replaced by the storage solution and concentrated to a specific concentration range, the tangential flow filtration system and the macroporous continuous chromatography system were used. The sample is collected and stored in the collection tube.

In the present invention, the buffer composition is determined according to the adsorption medium of macroporous continuous chromatography.

The crude protein solution to be purified in the present invention refers to the crude protein solution obtained after preliminary purification of the obtained fermented cells through centrifugation, cell disruption, centrifugation and filtration.

The storage solution of protein in the present invention refers to a solution for storing purified protein.

The macroporous continuous chromatography column in the present invention refers to a protein chromatography column based on macroporous continuous chromatography technology, that is, the chromatography column is a purification column that combines macroporous continuous chromatography technology with protein chromatography, such as a hydrophobic chromatography column, an affinity Chromatography column, molecular sieve, anion exchange chromatography column, cation exchange chromatography column, composite chromatography column, or other protein chromatography column based on macropore continuous chromatography technology. Beneficial effects of the present invention:

In the present invention, the tangential flow filtration device plays the role of concentration, buffer solution replacement and protein storage solution replacement, and the macroporous continuous chromatography device plays the role of purification. The tangential flow filtration system integrates the concentration of cell supernatant, buffer exchange, purification and formulation of the final product; at the same time, the system integrates the downstream purification steps, eliminating the need to replace the chromatographic column and readjust the conditions. The one-step operation from cell liquid recovery to final product purification can achieve the purpose of time-saving, labor-saving, economical and efficient purification. In addition, the system can further integrate ultraviolet light detection, pH, conductivity meter, etc. to realize real-time monitoring of the entire purification process, and finally establish an efficient, simple and economical laboratory and pilot-scale antibody production platform, which can be used in various protein purification pilot scales. The platform has great application prospects.

The system of the invention has better purification effect, such as processing monoclonal antibody protein, the content of host cell protein (HCP) in the cell fermentation broth before purification is 300,000 ppm, and the content of DNA is 30,000 ppm. After octanoic acid-allantoin-centrifugation-filtration pretreatment, the HCP content was reduced to less than 500ppm. After purification by a single-channel multiplexed convection chromatography system, the HCP content was less than 100ppm and the DNA content was less than 10ppm; the purity of IgG1 was over 95%, and the recovered rate is higher than 85%.

Description of drawings

Fig. 1 is the structural representation of single-channel multiplexed convection chromatography;

Fig. 2 is the purification principle diagram of single-channel multiplexed convection chromatography;

Fig. 3 is the structural representation of multi-channel multiplexing convection chromatography;

Fig. 4 is the purification principle diagram of multi-channel multiplexing convection chromatography;

In the figure: 1, feed storage tank; 2, peristaltic pump I; 3, tangential flow filter device; 4, circulating fluid storage tank; 5, peristaltic pump II; 6, peristaltic pump III; 7, waste filtrate storage liquid tank; 8, peristaltic pump IV; 9, macroporous continuous chromatography device; 10, pressure sensor; 11, weighing device; 12, pH and conductivity sensor; 13, peristaltic pump V.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

Example 1

As shown in FIG. 1, the present embodiment provides a single-channel multiplexing convection chromatography system including a feed liquid storage tank 1, a peristaltic pump I2, a tangential flow filter device 3, a circulating liquid storage tank 4, a peristaltic pump II5, Peristaltic pump III6, waste filtrate storage tank 7, peristaltic pump IV8, macroporous continuous chromatography device 9, peristaltic pump V13 and central processing unit; Wherein: described feed storage tank 1 is by peristaltic pump I2 and tangential flow filter device The feed ports of 3 are connected; the tangential flow filter device 3 is provided with two discharge ports, which are respectively a recovery filtrate discharge port and a waste filtrate discharge port; The filtrate storage tank 7 is connected; the reclaimed filtrate discharge port is divided into two branches, one branch is connected with the feed port of the circulating liquid storage tank 4 by the peristaltic pump V13, and the other branch is connected with the large by the peristaltic pump IV8. The total feed port of the continuous chromatographic device 9 is connected; the discharge port of the circulating liquid storage tank 4 is connected to the feed port of the tangential flow filter device 3 through the peristaltic pump II5; The total discharge port is connected with the feed port of the circulating liquid storage tank 4; the central processing unit controls the start and stop of the peristaltic pump I2, the peristaltic pump II5, the peristaltic pump III6, the peristaltic pump IV8 and the peristaltic pump V13.

The multiplexed convection chromatography system in this embodiment is a single-channel multiplexed convection chromatography system, wherein the macroporous continuous chromatography device 9 includes a macroporous continuous chromatography column. The macroporous continuous chromatography column is selected from any one of hydrophobic chromatography column, affinity chromatography column, molecular sieve, anion exchange chromatography column, cation exchange chromatography column and composite chromatography column. The tangential flow filtration device 3 comprises a tangential flow filtration column.

The feed storage tank 1 of this embodiment is used to store the crude protein solution, buffer solution and protein storage solution to be purified and processed respectively.

As a preferred way of this embodiment, on the pipeline between the peristaltic pump I2 and the feed port of the tangential flow filter device 3, on the pipeline between the peristaltic pump II5 and the feed port of the tangential flow filter device 3, Pressure sensors 10 are provided between the peristaltic pump IV8 and the total inlet of the macroporous continuous chromatography device 9 and between the waste filtrate outlet and the peristaltic pump III6; The collected pressure signal is sent to the central processing unit.

As a preferred mode of this embodiment, pH and conductivity sensors are provided between the peristaltic pump I2 and the feed storage tank 1 and between the peristaltic pump II5 and the discharge port of the circulating liquid storage tank 4 12; pH and conductivity sensor 12 is data-connected to the central processing unit, and the pH/conductivity sensor transmits the collected signals to the central processing unit.

As a preferred mode of this embodiment, the multiplexed convection chromatography system further includes a weighing device 11 , and the weighing device 11 is connected to the circulating liquid storage tank 4 for weighing the circulating liquid storage tank 4 the weight of the solution. The weighing device 11 can be an electronic scale, which directly measures the weight of the circulating liquid storage tank 4 and the solution stored therein; the weighing device 11 can also be a weight sensor, and the weight sensor collects the circulating liquid storage tank 4 and the stored solution. The weight information of the solution is sent to the central processing unit.

The technical schematic diagram of the single-channel multiplexing convection chromatography system of this embodiment is shown in FIG. 2 .

This embodiment also provides a method for purifying protein by utilizing the above-mentioned multiplexed convection chromatography system, the method comprising the following steps:

1) crude protein solution to be purified, such as monoclonal antibody cell fermentation broth, is stored in feed storage tank 1, starts peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 by central processing unit, Close the peristaltic pump IV8, the crude protein solution to be purified in the feed storage tank 1 is transported to the tangential flow filtration device 3 for filtration, and the waste filtrate is transported to the waste filtrate storage liquid by the peristaltic pump III6 in the filtration process In the tank 7, the recovered filtrate is transported to the circulating liquid storage tank 4, and after being weighed by the weighing device 11, it is transported back to the tangential flow filter device 3 by the peristaltic pump II5, and the above-mentioned filtering process is repeated to reach a predetermined concentration. one-stage concentration;

2) the solution in the feed storage tank 1 is replaced with a buffer, the buffer in the feed storage tank 1 is transported to the tangential flow filter device 3 by the peristaltic pump 12 to carry out buffer replacement, in the replacement process The waste liquid after the replacement is transported to the waste filtrate storage tank 7 by the peristaltic pump III6, the recovered liquid after the replacement is transported to the circulating liquid storage tank 4, and is transported back by the peristaltic pump II5 after being weighed by the weighing device 11. In the tangential flow filtration device 3, the above-mentioned replacement process is repeated to complete the buffer replacement to a predetermined pH and conductivity;

3) start peristaltic pump II5 and peristaltic pump IV8 by central processing unit, close peristaltic pump V13, make the solution in tangential flow filter device 3 and circulating liquid storage tank 4 enter macroporous continuous chromatographic device 9 and carry out impurity removal and purification, when The pollutant adsorption approaches equilibrium, the macroporous continuous chromatography is offline, and the solution after the impurity removal by the macroporous continuous chromatography device 9 enters the circulating liquid storage tank 4 for storage;

4) start peristaltic pump I2, peristaltic pump II5, peristaltic pump III6 and peristaltic pump V13 by central processing unit, close peristaltic pump IV8, the solution in the feed storage tank 1 is replaced by the storage solution of protein, the circulating fluid storage solution The solution stored in the tank 4 after the impurity removal by the macroporous continuous chromatography device 9 is transported to the tangential flow filtration device 3 for repeated circulation treatment to complete the secondary concentration and replacement of the protein storage liquid, and the final treated liquid is replaced by the circulating liquid. Storage tank 4 is stored;

5) when monoclonal antibody concentration reaches 1mg/mL, close peristaltic pump I2, peristaltic pump II5, peristaltic pump III6, peristaltic pump V13 and peristaltic pump IV8 by central processing unit, collect the solution in circulating fluid storage tank 4, namely A purified protein solution was obtained.

Example 2

As shown in FIG. 3 , the difference between this embodiment and Embodiment 1 is that the multiplexed convection chromatography system is a multi-channel multiplexed convection chromatography system, wherein the macroporous continuous chromatography device 9 includes two macroporous continuous chromatography columns connected in parallel. The two macroporous continuous chromatographic columns connected in parallel are an anion exchange chromatographic column and a hydrophobic chromatographic column, respectively. It can also be any combination selected from hydrophobic chromatography columns, affinity chromatography columns, molecular sieves, anion exchange chromatography columns, cation exchange chromatography columns and composite chromatography columns.

The technical schematic diagram of the multi-channel multiplexing convection chromatography system of this embodiment is shown in FIG. 4 .

The difference between the purification method in this example and Example 1 is that when the monoclonal antibody concentration reaches 5 mg/mL, the TFF system goes offline, the sample processing ends, and the final purified sample is collected and stored in a collection tube.

Example 3

Taking the monoclonal antibody IgG1 as an example, it shows that the single-channel multiplexed convection chromatography system can complete the one-step operation from concentration, recovery to purification of cell fermentation broth.

B搅拌式玻璃生物反应器中生产，采用分批补料的培养方式，培养15～30天后收集细胞发酵液。细胞培养基为无蛋白培养基CD CHO(LifeTechnologies)和无蛋白培养基

PF-CHO(GE Healthcare)的混合物，两者体积比为1:1。Cell culture: Monoclonal antibody IgG1 was expressed by Chinese Hamster Ovary cells (Chinese Hamster Ovary, CHO).

B is produced in a stirred glass bioreactor, and the culture method of fed-batch is adopted, and the cell fermentation broth is collected after culturing for 15 to 30 days. Cell culture medium is protein-free medium CD CHO (LifeTechnologies) and protein-free medium

A mixture of PF-CHO (GE Healthcare) in a volume ratio of 1:1.

Rapid-Flow Filters,Thermo Scientific)过滤，收集过滤液。Cell fermentation broth pretreatment: the pH of the cell fermentation broth was adjusted to 5.3, and caprylic acid and allantoin were added to make the final mass concentrations of caprylic acid and allantoin in the above mixture 0.4% and 1%, respectively. The mixture was stirred at room temperature for 2 hours, then centrifuged at 4000 × g for 20 min at room temperature, and the supernatant was collected through a 0.22 μm filter (

Rapid-Flow Filters, Thermo Scientific) to collect the filtrate.

Filtrate (about 5 liters) is placed in feed storage tank 1, enters tangential flow filter device 3 (tangential flow filter column, TFF) via peristaltic pump 1, discards filtrate through peristaltic pump III6 discards filtrate storage tank 7 After being discharged from the system, the sample concentrated through the tangential flow filter device 3 enters the circulating liquid storage tank 4 for weighing and then enters the TFF column again for concentration. When the circulating fluid volume was concentrated to about 3 liters, Tris-HCl buffer (pH 7.5) was added to the feed storage tank 1, and the Tris-HCl buffer entered the TFF column for buffer exchange of the concentrated sample. After the circulating fluid is replaced by the buffer, the central processing unit controls the macroporous continuous chromatographic device 9 (single-channel multiplexing convection chromatography system, the macroporous continuous chromatographic column adopts an anion exchange chromatography column). The pore continuous chromatography device 9 (anion exchange column) starts IgG1 purification, and after all the circulating liquid flows out from the macropore continuous chromatography device 9 (anion exchange column), the purification is completed and the AMC system goes offline. After purification, IgG1 entered the TFF system for further concentration, and at the same time, PBS buffer (pH 7.2-7.4, 10 mM) was added to the feed storage tank 1 as a monoclonal antibody storage solution to replace the storage solution for IgG1. When the IgG1 concentration reaches 3 mg/mL, the TFF system goes offline, the sample processing ends, and the final purified sample is collected and stored in the collection tube. The host protein content was detected by Generation III CHO HCP kit, and the DNA content was detected by digital PCR instrument QX100 ^™ Droplet Digital ^™ PCR System (Bio-Rad).

Experimental results: In the cell fermentation broth before purification, the content of host cell protein (HCP) was 300,000 ppm, and the content of DNA was 30,000 ppm. After octanoic acid-allantoin-centrifugation-filtration pretreatment, the HCP content was reduced to below 500ppm. After being purified by a single-channel multiplexed convection chromatography system, the content of HCP is less than 100 ppm, and the content of DNA is less than 10 ppm; the purity of IgG1 is over 95%, and the recovery rate is higher than 85%. Comply with the existing U.S. Food and Drug Administration (FDA) requirements for monoclonal antibody protein products.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the 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.

Images