CN110923366A

CN110923366A - Method for on-line monitoring yield and quality of target protein

Info

Publication number: CN110923366A
Application number: CN202010086190.9A
Authority: CN
Inventors: 谢红伟; 李思鹏; 刘卓裕
Original assignee: Shanghai Fu Hong Han Lin Bio Pharmaceutical Co Ltd; Shanghai Fuhong Hanlin Biomedical Co Ltd; Shanghai Henlius Biotech Inc
Current assignee: Shanghai Fu Hong Han Lin Bio Pharmaceutical Co Ltd; Shanghai Fuhong Hanlin Biomedical Co Ltd; Shanghai Henlius Biotech Inc
Priority date: 2020-02-11
Filing date: 2020-02-11
Publication date: 2020-03-27

Abstract

The invention is suitable for the technical field of biology, and provides a method for monitoring the yield and quality of a target protein on line, wherein a first dimension chromatographic separation device receives a control signal of a control device, performs protein affinity chromatographic separation on cell culture fermentation broth containing the target protein to obtain eluent containing the target protein, and returns a first detection signal to the control device; the control device calculates the yield information of the target protein solution according to the first detection signal; the second dimension chromatographic separation device receives the control signal of the control device, and carries out cation exchange chromatography and/or size exclusion chromatographic separation on the eluent containing the target protein to obtain a target protein solution, and returns a second detection signal to the control device; and the control device calculates the quality information of the target protein solution according to the second detection signal. Therefore, the invention realizes automatic monitoring of the yield and quality of the target protein solution and improves the monitoring efficiency.

Description

Method for on-line monitoring yield and quality of target protein

Technical Field

The invention relates to the technical field of biology, in particular to a method for monitoring yield and quality of target protein on line.

Background

With the continuous improvement of the production process of monoclonal antibodies (abbreviated as mabs), the concept of quality origin from design is also gradually applied to various stages of production process and process optimization. Therefore, it is imperative to monitor the yield and key quality attributes of the monoclonal antibody product in real time during the cell culture phase. In the cell culture stage, if Critical Quality Attributes (CQA) such as yield, purity, glycoform and the like need to be monitored simultaneously, a corresponding analysis method is required to support; the components in the sample at this stage are complex, and if the analysis of purity or other quality attributes is required, one-step manual purification is required, which is time-consuming and labor-consuming, and the purification operation steps are difficult to verify; meanwhile, the process waiting time is increased; and the stability of the early sample is poor, which directly influences the accuracy of the analysis method.

In recent years, more and more biological products, especially monoclonal antibody products, enter the commercial production stage, and the corresponding production process is moving towards automation and continuous development, which demands higher requirements on monitoring of the production process and timeliness of result feedback. Therefore, the importance of a multi-attribute online monitoring technology to an automatic production process is self-evident.

The two-dimensional liquid chromatography is a research hotspot in the field of the current liquid chromatography, focuses on the application of characteristic analysis, component identification, structural characterization, online purification and the like of products such as proteins, polypeptides, small-molecule drugs and the like, is two sets of liquid chromatography connected by a plurality of valve components, and can greatly improve the feasibility of simultaneously analyzing various product attributes. However, the application and development of the two-dimensional liquid phase are mostly seen in the research and development stage, and the research aiming at the two-dimensional liquid phase is mostly focused on the updating and optimization of the liquid phase component, and there is only a few attempts to combine the two-dimensional liquid phase component into the production process flow.

In view of the above, the prior art is obviously inconvenient and disadvantageous in practical use, and needs to be improved.

Disclosure of Invention

In view of the above-mentioned drawbacks, the present invention provides a method for on-line monitoring the yield and quality of a target protein, so as to automatically monitor the yield and quality of a target protein solution and improve the monitoring efficiency.

In order to achieve an object of the present invention, the present invention provides a method for online monitoring of yield and quality of a target protein, which is implemented using a system for online monitoring of yield and quality of a target protein; the system comprises: the device comprises a control device, a first dimension chromatographic separation device and a second dimension chromatographic separation device; the method comprises the following steps:

A. the first dimension chromatographic separation device receives the control signal of the control device, performs protein A affinity chromatographic separation on the cell culture fermentation broth containing the target protein to obtain an eluent containing the target protein, and returns a first detection signal to the control device; the control device calculates the yield information of the target protein solution according to the first detection signal;

B. the second dimension chromatographic separation device receives the control signal of the control device, and carries out cation exchange chromatography and/or size exclusion chromatographic separation on the eluent containing the target protein to obtain a target protein solution, and returns a second detection signal to the control device; and the control device calculates the quality information of the target protein solution according to the second detection signal.

According to the method, the system further comprises: a cell culture sampling device; the method further comprises the following steps:

C. the cell culture sampling device receives the control signal of the control device, samples a cell culture sample containing the target protein, and centrifuges and/or filters the cell culture sample to obtain the cell culture fermentation liquor containing the target protein.

According to the method, the first dimension chromatographic separation device comprises a first sample injection module, a Protein A affinity chromatographic column and a first detector;

the first sample injection module comprises a sample injection ring and a collector, and the sample injection ring is connected with the cell culture sampling device and the collector through a first multi-angle valve;

the first detector is connected with the Protein A affinity chromatographic column and the collector;

the control device includes: the device comprises a first control module, a second control module, a third control module and a first calculation module;

the step A comprises the following steps:

a1, the first control module sends a first control signal to the first sample injection module, and the sample injection loop is controlled to convey the cell culture fermentation broth containing the target Protein to the Protein A affinity chromatographic column;

a2, the second control module sends a third control signal to the Protein A affinity chromatographic column, and the Protein A affinity chromatographic column is controlled to carry out Protein elution on the cell culture fermentation broth containing the target Protein, so as to obtain an eluent containing the target Protein;

a3, the third control module sends a fourth control signal to the first detector, and the first detector returns the first detection signal;

a4, the first calculation module calculates the yield of the target protein solution according to the first detection signal.

According to the method, the second dimension chromatographic separation device comprises a cation exchange chromatographic column, a size exclusion chromatographic column and a second detector; the Protein A affinity chromatographic column, the cation exchange chromatographic column and the size exclusion chromatographic column are connected with the collector through a second multi-angle valve;

the control device further includes: the device comprises a series path selection module, a fifth control module and a second calculation module;

the step B comprises the following steps:

b1, the serial path selection module controls the path of the second multi-angle valve, and eluent containing the target protein is arranged to pass through the cation exchange chromatographic column or the size exclusion chromatographic column;

b2, the cation exchange chromatographic column is used for carrying out cation exchange chromatographic separation on the eluent containing the target protein; or the size exclusion chromatography column performs size exclusion chromatography on the eluent containing the target protein; obtaining the target protein solution;

b3, sending a fifth control signal to the second detector by the fifth control module, controlling the second detector to detect an ultraviolet absorption signal of the eluent containing the target protein passing through the cation exchange chromatographic column or the size exclusion chromatographic column, and returning the second detection signal to the control device;

b4, the second calculation module calculates the charge heterogeneity or monomer purity and molecular size heterogeneity of the target protein solution product according to the second detection signal.

According to the method, the second dimension chromatographic separation device comprises a cation exchange chromatographic column and a size exclusion chromatographic column; the Protein A affinity chromatography column, the cation exchange chromatography column, or the Protein A affinity chromatography column and the size exclusion chromatography column are connected with the first detector through a third multi-angle valve;

a second detector is connected to the cation exchange chromatography column or the size exclusion chromatography column;

the control device further includes: the parallel path selection module, the fifth control module and the second calculation module;

the step B comprises the following steps:

b5, the parallel channel selection module controls the channel of the third multi-angle valve to arrange the eluent containing the target protein to pass through the cation exchange chromatographic column and the size exclusion chromatographic column;

b6, respectively carrying out cation exchange chromatography separation and size exclusion chromatography separation on the eluent containing the target protein by the cation exchange chromatography column and the size exclusion chromatography column to obtain the target protein solution;

b7, the fifth control module sends a sixth control signal to the second detector and the first detector, detects the ultraviolet absorption signal of the eluent containing the target protein passing through the cation exchange chromatographic column and the size exclusion chromatographic column, and returns the second detection signal to the control device;

b8, the second calculation module calculates the charge heterogeneity, monomer purity and molecular size heterogeneity of the target protein solution according to the second detection signal.

According to the method, the cell culture sampling device comprises a cell culture tank, a centrifuge and/or a filter which are connected with each other; the control device further includes: a fourth control module;

the step C comprises the following steps:

c1, the fourth control module controls the cell culture tank to culture a cell culture sample containing the target protein and deliver to the centrifuge and/or filter;

c2, the fourth control module controls the centrifuge and/or the filter to centrifuge and/or filter the cell culture sample containing the target protein to obtain the cell culture fermentation liquid containing the target protein.

According to the method, the system further comprises: the first pump module is connected with the first sample introduction module and the second dimensional chromatographic separation device, and the waste liquid recoverer is connected with the second dimensional chromatographic separation device;

the method further comprises the following steps:

the first pump module provides power for mobile phase, isocratic or gradient elution when the first dimension chromatographic separation device and the second dimension chromatographic separation device carry out chromatographic separation; and/or

And the second dimension chromatographic separation device conveys the waste liquid generated after the chromatographic separation to the waste liquid recoverer.

According to the method, the Protein A affinity chromatographic column, the cation exchange chromatographic column and the size exclusion chromatographic column are arranged in a multi-column temperature box;

the first detector and the second detector are both ultraviolet detectors;

the first pump module comprises a left pump and a right pump; the left pump is connected with the Protein A affinity chromatographic column, and the right pump is connected with the cation exchange chromatographic column and/or the size exclusion chromatographic column;

the multi-angle valve is a hexagonal valve.

According to the method, the cell culture sample is a cell culture sample comprising a protein of interest.

The invention provides a method for monitoring the yield and the quality of target protein on line by using a system for monitoring the yield and the quality of the target protein on line, which comprises a control device, a first dimension chromatographic separation device and a second dimension chromatographic separation device, wherein the first dimension chromatographic separation device and the second dimension chromatographic separation device form a two-dimension liquid phase system, and the two-dimension liquid phase system is connected to a cell culture sampling device for real-time sampling in the cell culture process, and is directly connected to a two-dimension liquid phase for analyzing the yield and the quality (the charge heterogeneity or the purity and the molecular size heterogeneity) after being connected to a cell culture sampling device for real-time sampling, so that the yield and the quality of fermentation liquor at the upstream of a target protein solution are automatically monitored, and the monitoring efficiency is improved.

Drawings

FIG. 1 is a block diagram of a system for online monitoring of yield and quality of a fermentation broth upstream of a monoclonal antibody according to an embodiment of the present invention;

FIG. 2 is a block diagram of a system for online monitoring of yield and quality of a target protein according to an embodiment of the present invention;

FIG. 3 is a block diagram of a system for online monitoring of yield and quality of a target protein according to an embodiment of the present invention;

FIG. 4 is a schematic flow chart of online monitoring of yield and quality of fermentation broth upstream of a target protein according to an embodiment of the present invention;

FIG. 5 is a schematic flow chart of online monitoring of yield and quality of fermentation broth upstream of a target protein according to an embodiment of the present invention;

FIG. 6 is a flow chart of a method for on-line monitoring of yield and quality of fermentation broth upstream of a protein of interest according to an embodiment of the present invention;

FIG. 7 is a Protein A-CEX spectrum in data analysis for on-line monitoring of yield and quality of fermentation broth upstream of a Protein of interest provided by an embodiment of the present invention;

FIG. 8 is a Protein A-SEC profile in data analysis for online monitoring of yield and quality of fermentation broth upstream of a Protein of interest provided by an embodiment of the present invention;

FIG. 9 is a schematic diagram of the polygon valve switching in a system for online monitoring of yield and quality of fermentation broth upstream of a target protein according to an embodiment of the present invention;

FIG. 10 is a schematic diagram of polygon valve switching in a system for online monitoring of yield and quality of fermentation broth upstream of a target protein according to an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Material sources are as follows:

test sample mab 1 was a recombinant human-mouse chimeric anti-CD 20 monoclonal antibody (Rituximab biosimilar).

Test sample mab 2 was a recombinant anti-CD 38 humanized monoclonal antibody (Daratumumab biosimilar).

Referring to fig. 1-5, in one embodiment of the present invention, a system 100 for online monitoring of yield and quality of a target protein is provided, comprising:

a control device 10 for executing an on-line monitoring operation instruction, outputting a control signal, and calculating information on yield and quality related to the target protein according to the received detection signal; the control device 10 executes a preset or on-site control online monitoring operation instruction, and sends corresponding control signals to a plurality of devices in the system 100 to control the plurality of devices to execute corresponding operations;

the first dimension chromatographic separation device 20 is configured to receive the control signal from the control device 10, perform Protein a affinity chromatographic separation on the cell culture fermentation broth containing the target Protein to obtain an eluate containing the target Protein, and return a first detection signal to the control device 10; the first dimension chromatographic separation device 20 is used for carrying out Protein A affinity chromatographic separation on the cell culture fermentation liquor containing the target Protein;

a second dimension chromatographic separation device 30, configured to receive the control signal from the control device 10, perform cation exchange chromatography and/or size exclusion chromatography on the eluate containing the target protein to obtain a target protein solution, and return a second detection signal to the control device; the second dimension chromatographic separation device 30 is used for carrying out cation exchange chromatography and/or size exclusion chromatographic separation on eluent containing the target protein;

wherein the control device 10 calculates the yield information of the target protein solution according to the first detection signal; and calculating the quality information of the target protein solution according to the second detection signal.

In this example, the conventional Protein A affinity chromatography and cation exchange Chromatography (CEX) and/or Size Exclusion Chromatography (SEC) are linked by a two-dimensional liquid phase switching valve, and the monitoring result is consistent with the single analysis result, so that the manual operation time is greatly saved, such as shortening from 1-2 days of off-line monitoring to 1 hour of on-line monitoring, and the on-line monitoring of the single antibody yield and the quality attribute in the cell culture process is realized. Moreover, the method is particularly suitable for monoclonal antibody process development and online central control detection of upstream fermentation liquor. Wherein the cell culture sample is a monoclonal antibody cell culture sample. Furthermore, the protein solution of interest may be any protein solution, in particular any recombinant protein produced by culturing a host cell. The most preferred target protein solutions are antibodies (monoclonal antibodies), immunoadhesins and other antibody-like molecules, such as fusion proteins comprising the CH2/CH3 region. In the illustrations of fig. 4 and 5, the first-dimension liquid-phase flow path represents the liquid-phase flow path of the first-dimension chromatographic separation device 20; the second-dimension liquid phase flow path represents a liquid phase flow path of the second-dimension chromatographic separation device 30.

Referring to fig. 1-5, in one embodiment of the present invention, the system 100 for online monitoring of yield and quality of a target protein further comprises:

and the cell culture sampling device 40 is used for receiving the control signal of the control device 10, sampling a cell culture sample containing the target protein, centrifuging and/or filtering the cell culture sample to obtain cell culture fermentation liquor containing the target protein. The cell culture sampling device 40 samples, centrifuges and/or filters a cell culture sample containing a target protein to obtain a cell culture fermentation broth containing the target protein.

the first pump module 50 is connected with the first sample feeding module 21 and the second dimension chromatographic separation device 30, and the first pump module 50 is used for providing power for mobile phase, isocratic or gradient elution for the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30; and the first pump module 50 includes a left pump 51 and a right pump 52; the left pump 51 is connected with the first dimension chromatographic separation device 20, the right pump 52 is connected with the second dimension chromatographic separation device 30, and the mobile phase, the isocratic or gradient elution power for chromatographic separation are respectively provided for the left pump and the right pump.

In addition, the system 100 for on-line monitoring the yield and quality of the target protein further comprises a waste liquid recoverer 60 connected with the second dimension chromatographic separation device 30, wherein the waste liquid recoverer 60 is used for receiving waste liquid generated after the separation by the second dimension chromatographic separation device 30.

Referring to fig. 1-5, in one embodiment of the present invention, a first dimension chromatographic separation device 20 comprises:

the first sample injection module 21 comprises a sample injection ring 311 and a collector 312, wherein the sample injection ring 311 is connected with the cell culture sampling device 40 and the collector 312 through a first multi-angle valve 46; the first sample injection module 21 can automatically transmit the cell culture fermentation broth containing the target Protein, which is cultured, sampled, centrifuged and/or filtered by the cell culture sampling device 40, to the Protein A affinity chromatography column 22; the cell culture sampling device 40 includes a cell culture tank 41, a centrifuge and/or a filter 42 connected to each other.

A Protein A affinity chromatographic column 22 for eluting the Protein of the cell culture fermentation broth containing the target Protein to obtain an eluate containing the target Protein; the Protein in the cell culture fermentation broth containing the target Protein is captured by the Protein a affinity column 22, and the Protein is eluted from the Protein a affinity column 22 by the elution phase through the control device 10.

And a first detector 23 connected to the Protein a affinity chromatography column 22 and the collector 312, wherein the first detector 23 is used for detecting a first detection signal of the ultraviolet absorption signal of the eluent containing the target Protein flowing through. The first detector 23 captures an ultraviolet absorption signal of the protein of the eluent containing the target protein flowing through, and returns a first detection signal containing information of the ultraviolet absorption signal to the control device 10, and the first detection signal is used for calculating the yield of the target protein solution and the content of the second dimension chromatogram sample.

Referring to fig. 3 to 5, in one embodiment of the present invention, the control device 10 includes:

the first control module 11 is configured to send a first control signal to the first sample injection module 21, and control the sample injection loop 311 to deliver the cell culture fermentation broth containing the target Protein to the Protein a affinity chromatography column 22; and

for sending a second control signal to the collector 312, recovering the eluate containing the target protein detected by the first detector 23;

the second control module 12 is configured to send a third control signal to the Protein a affinity chromatography column 22, and control the Protein a affinity chromatography column 22 to perform Protein elution on the cell culture fermentation broth containing the target Protein, so as to obtain an eluate containing the target Protein;

a third control module 13, configured to send a fourth control signal to the first detector 23, and control the first detector 23 to return the first detection signal; the first detector 23 returns a first detection signal of an ultraviolet absorption signal of the eluent which is passed through and contains the target protein;

a first calculating module 15, configured to calculate a yield of the target protein solution according to the first detection signal. The first detection signal is an ultraviolet absorption signal including an eluent containing the target protein, and the eluent is calculated by the first calculation module 15 to obtain the yield of the target protein solution and the sample amount of the second dimension chromatogram.

In one embodiment of the present invention, the control device 10 further includes:

and the fourth control module 14 is configured to send a seventh control signal to the cell culture sampling device 40, control the cell culture sampling device 40 to culture a cell culture sample containing a target protein, perform centrifugation and/or filtration, and then convey the obtained cell culture fermentation broth containing the target protein to the first sample injection module 21. The cell culture sampling device 40 is controlled by the fourth control module 14 to culture the cell culture sample and to centrifuge and/or filter the cell culture sample.

Referring to fig. 1-3, and 4, in one embodiment of the present invention, a second dimension chromatographic separation device 30 comprises:

a cation exchange chromatography column 31 for subjecting the eluate containing the target protein to cation exchange chromatography;

a size exclusion chromatography column 32 for size exclusion chromatographic separation of the eluate containing the target protein;

the Protein a affinity chromatography column 22, cation exchange chromatography column 31 or size exclusion chromatography column 32 is connected to the collector 312 by a second multi-angle valve 47; after the second multi-angle valve 47 is controlled by the control device 10 to set the cell culture fermentation broth containing the target Protein to pass through the Protein A affinity chromatography column 22, the eluent containing the target Protein passes through the cation exchange chromatography column 31 or the size exclusion chromatography column 32;

a second detector 33 connected to the cation exchange chromatography column 31 and the size exclusion chromatography column 32; the second detector 33 is configured to detect the ultraviolet absorption signal of the target protein solution flowing through, and return the second detection signal containing the ultraviolet absorption signal of the target protein solution to the control device 10.

In this embodiment, the control device 10 includes:

a serial path selection module 16 for arranging an eluate containing the target protein through the cation exchange chromatography column 31 or size exclusion chromatography column 32 for passage through the control second multi-angle valve 47; after the serial path selection module 16 controls the second multi-angle valve 47 to open the path of the eluent containing the target protein through the cation exchange column 31, the path of the other eluent containing the target protein through the size exclusion column 32 is closed, and vice versa, that is, the eluent containing the target protein in this embodiment only passes through one of the cation exchange column 31 and the size exclusion column 32 according to the control.

A fifth control module 17, configured to send a fifth control signal to the second detector 33, detect a second detection signal of the ultraviolet absorption signal of the eluent containing the target protein passing through the cation exchange chromatography column 31 or the size exclusion chromatography column 32, return the second detection signal to the control device 10, and obtain the target protein solution;

and a second calculating module 18, configured to calculate a charge heterogeneity or a monomer purity and a molecular size heterogeneity of the target protein solution according to the second detection signal. In this embodiment, the eluent containing the target protein is selected by the serial path selection module 16 to pass through the cation exchange chromatography column 31 or the size exclusion chromatography column 32, so that the charge heterogeneity of the target protein solution, or the monomer purity and molecular size heterogeneity can be calculated. The information of the mass of different target protein solutions can be obtained according to different selected pathways, and can be the charge heterogeneity of the target protein solutions or the monomer purity and molecular size heterogeneity of the target protein solutions.

Referring to fig. 1-3 and 5, in one embodiment of the present invention, the second dimension chromatographic separation device 30 comprises:

the Protein A affinity chromatographic column 22, the cation exchange chromatographic column 31, or the Protein A affinity chromatographic column 22 and the size exclusion chromatographic column 32 are connected with the first detector 23 through a third multi-angle valve 48;

the cation exchange chromatography column 31 or the size exclusion chromatography column 32 is connected to the second detector 33;

the first detector 23 is further configured to detect a second detection signal of the ultraviolet absorption signal of the eluent of the target protein flowing through, and return the first detection signal to the control device 10. In this embodiment, the first detector 23 undertakes detection of a second detection signal of the second dimension chromatographic separation device 30 in addition to the aforementioned return of the first detection signal to return the second detection signal to the control device 10.

Unlike the previous embodiment, in this embodiment, the cation exchange column 31 and the size exclusion column 32 belong to parallel paths, and the eluent containing the target protein flows through the cation exchange column 31 and the size exclusion column 32 simultaneously; the control device 10 is used for controlling, and the specific control device 10 comprises:

a parallel path selection module 19 for setting the eluent containing the target protein to pass through the cation exchange chromatography column 31 and the size exclusion chromatography column 32 by controlling the path of the third multi-angle valve 48;

a fifth control module 17, configured to send a sixth control signal to the second detector 33 and the first detector 23, detect an eluate containing the target protein passing through the cation exchange chromatography column 31 and the size exclusion chromatography column 32, and obtain the target protein solution; in the case where the eluate containing the target protein passes through both the cation exchange chromatography column 31 and the size exclusion chromatography column 32, the first detector 23 will also take part in the detection of a portion of the second detection signal of the second dimensional chromatographic separation device 30;

a second calculating module 18, configured to calculate charge heterogeneity, purity and molecular size heterogeneity of the target protein solution product according to the second detection signal;

in this embodiment, the multi-angle valve is controlled by the parallel channel selection module 19 to pass the eluates containing the target protein through the cation exchange column 31 and the size exclusion column 32; the second calculation module 18 calculates and obtains the charge heterogeneity, monomer purity and molecular size heterogeneity of the target protein solution. The fourth multi-angle valve 49 in fig. 5 is connected to the first and

second detectors

23 and 33, respectively, and the collector 312.

In addition, the preferred Protein A affinity column 22, cation exchange column 31 and size exclusion column 32 are disposed in a multi-column incubator 45; the system 100 for monitoring the yield and quality of the target protein on line has a more compact structure and is convenient to operate. The first detector 23 and the second detector 33 are both ultraviolet detectors, and return a first detection signal and a second detection signal. In the above embodiments, the multi-angle valve is a six-angle valve, that is, the first to fourth multi-angle valves may be a six-angle valve or an eight-angle valve; and the cell culture sample is a cell culture sample comprising the protein of interest. In addition, each control module of the control device 10 may directly receive the feedback signal, or the control device 10 may further include a signal transmission module, which is configured to receive signals transmitted by other devices, such as the first detection signal, the second detection signal, and the like, and transmit the signals to each control module of the control device 10. The numbers of the first to seventh control signals described in the present invention do not represent the order of occurrence of the absolute control signals, but are only control functions for distinguishing the control signals sent by the plurality of modules of the control device 10.

In order to achieve another object of the present invention, the present invention provides a method for online monitoring of yield and quality of a target protein, which is implemented using the system 100 for online monitoring of yield and quality of a target protein; the system 100 includes: a control device 10, a first dimension chromatographic separation device 20 and a second dimension chromatographic separation device 30; the method comprises the following steps:

step S601, the first dimension chromatography separation device 20 receives the control signal of the control device 10, performs protein affinity chromatography separation on the cell culture fermentation broth containing the target protein to obtain an eluate containing the target protein, and returns a first detection signal to the control device 10; the control device 10 calculates the yield information of the target protein solution according to the first detection signal;

step S602, the second dimension chromatography separation device 30 receives the control signal from the control device 10, performs cation exchange chromatography and/or size exclusion chromatography on the eluate containing the target protein to obtain a target protein solution, and returns a second detection signal to the control device 10; the control device 10 calculates the quality information of the target protein solution according to the second detection signal.

In this embodiment, the system 100 for online monitoring of the yield and quality of the target Protein in the above embodiments is used to realize online monitoring of the yield and quality of the target Protein, specifically, the control device 10 controls the cell culture fermentation broth containing the target Protein to pass through Protein a affinity chromatography of the first dimension chromatography separation device 20 to obtain an eluate containing the target Protein, and the control device 10 calculates the yield information of the target Protein solution according to the received first detection signal; such as to obtain the concentration of its target protein solution (Titer); then, through central cutting, antibody peaks in Protein a affinity chromatography are collected, and enter cation exchange chromatography and/or size exclusion chromatography of a second dimension chromatography separation device 30, and the control device 10 analyzes the charge heterogeneity and/or purity and molecular size heterogeneity of the target Protein solution according to the received second detection signal; the whole process is automatically completed by the control device 10 according to the set program without manual intervention.

In one embodiment of the present invention, the first dimension chromatographic separation device 20 comprises a first sample injection module, a Protein A affinity chromatographic column 22 and a first detector 23;

the first sample injection module 21 comprises a sample injection loop 311 and a collector 312, wherein the sample injection loop 311 is connected with the cell culture sampling device 40 and the collector 312 through a first multi-angle valve 46;

the first detector 23 is connected to the Protein a affinity chromatography column 22 and the collector 312;

the control device 10 includes: a first control module 11, a second control module 12, a third control module 13 and a first calculation module 15;

the step S601 includes:

a1, the first control module 11 sends a first control signal to the first sample module 21, and controls the sample loop 311 to deliver the cell culture fermentation broth containing the target Protein to the Protein A affinity chromatography column 22;

a2 and the second control module 12 send a third control signal to the Protein A affinity chromatography column 22, and control the Protein A affinity chromatography column 22 to perform Protein elution on the cell culture fermentation broth containing the target Protein, so as to obtain an eluent containing the target Protein;

a3, the third control module 13 sends a fourth control signal to the first detector 23, and the first detector 23 returns the first detection signal;

a4, the first calculating module 15 calculates the yield of the target protein solution product according to the first detection signal.

Referring to FIGS. 1-3, as well as FIG. 4, in one embodiment of the present invention, the second dimension chromatographic separation device 30 comprises a cation exchange chromatography column 31 and/or a size exclusion chromatography column 32; the Protein a affinity chromatography column 22, cation exchange chromatography column 31 and size exclusion chromatography column 32 are connected to the collector 312 by a second multi-angle valve 47; the control device 10 further includes: a series path selection module 16, a fifth control module 17 and a second calculation module 18; the step S602 includes:

b1, the serial path selection module 16 controls the path of the second multi-angle valve 47, and an eluent containing the target protein is arranged to pass through the cation exchange chromatography column 31 or the size exclusion chromatography column 32;

b2, the cation exchange chromatographic column 31 carries out cation exchange chromatographic separation on the eluent containing the target protein; or the size exclusion chromatography column 32 performs size exclusion chromatography on the eluate containing the target protein; obtaining the target protein solution;

b3, sending a fifth control signal to the second detector 33 by the fifth control module 17, detecting the ultraviolet absorption signal eluent containing the target protein eluent passing through the cation exchange chromatography column 31 or the size exclusion chromatography column 32, and returning the second detection signal to the control device 10;

b4, the second calculating module 18 calculates the charge heterogeneity or monomer purity and molecular size heterogeneity of the target protein solution according to the second detection signal.

In this example, for the two-dimensional liquid phase constituted by the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30, Protein a affinity chromatography and SEC or CEX are respectively accessed, and a sample is collected by an autosampler. After the cell culture sample containing the target Protein is taken out from the cell culture tank 41, the cell culture sample flows into the sampling ring 311 of the two-dimensional liquid phase sampling system after centrifugal filtration, then the target Protein in the cell culture fermentation liquid containing the target Protein is captured onto a chromatographic column through the first dimension chromatographic separation device 20 and the first dimension Protein A affinity chromatography, and the target Protein is eluted from the chromatographic column by adopting an elution phase through the setting of the control device 10, so as to obtain an eluent containing the target Protein. The control device 10 may be implemented as a computer control system. Then, the sample flows through the first detector 23, the first detector 23 returns a first detection signal of the detected ultraviolet absorption signal of the target protein eluent to the control device 10, the control device 10 calculates the yield information of the target protein-containing eluent according to the fed back first detection signal, the information is fed back to the control device 10 in the form of the first detection signal, and the control device 10 controls the sample to be collected and returned to the collector 312 of the sample injection system; then, the sample collected by the first dimension chromatography separation device 20, that is, the eluate containing the target protein, is taken out by the first sample injection module 21 and is injected into the second dimension chromatography separation device 30 (CEX or SEC), the target protein solution is obtained by separation of the cation exchange chromatography column 31 or the size exclusion chromatography column 32, the second detector 33 returns the second detection signal of the detected ultraviolet absorption signal of the eluate of the target protein solution to the control device 10, and the control device 10 calculates the mass information of the charge heterogeneity or the monomer purity and the molecular size heterogeneity of the target protein solution according to the fed back second detection signal.

In one embodiment of the present invention, the system 100 further comprises: a cell culture sampling device 40; before step S601, the method further includes:

C. the cell culture sampling device 40 receives the control signal of the control device 10, samples the cell culture sample containing the target protein, and centrifuges and/or filters the cell culture sample to obtain the cell culture fermentation liquor containing the target protein. By this step, a cell culture sample can be sampled and processed by centrifugation and/or filtration.

The cell culture sampling device 40 comprises a cell culture tank 41, a centrifuge and/or a filter 42 connected to each other; the control device 10 further includes: a fourth control module 15;

specifically, step C comprises

Step C1, the fourth control module 15 controls the cell culture tank 41 to culture the cell culture sample containing the target protein and deliver the cell culture sample to the centrifuge and/or the filter 42;

step C2, the fourth control module 15 controls the centrifuge and/or the filter 42 to centrifuge and/or filter the cell culture sample containing the target protein, and then obtains the cell culture fermentation broth containing the target protein. The whole system 100 is thus fully automated from the preparation of the cell culture sample containing the protein of interest to the final acquisition of yield and quality information of said protein of interest solution.

Referring to fig. 4, in an embodiment of the present invention, according to the above-mentioned operation of passing the cell culture broth through the cation exchange chromatography column 31 or the size exclusion chromatography column 32, the following steps (one) to (three) are required before the step C;

step (I): reagent preparation (Protein A affinity chromatography and cation exchange two-dimensional chromatography are taken as examples). The preparation of the reagent can be carried out according to the times described below, and the content of the components of the reagent is ensured to be unchanged.

A hydrochloric acid solution (1.2 mol/L), a sodium hydroxide solution (4.0 mol/L), a Protein A mobile phase A (10.0 mmol/L phosphate, 2.7 mmol/L KCl and 137.0 mmol/L NaCl, pH 7.4), a Protein A mobile phase B (12.0 mmol/L HCl, 150.0 mmol/L NaCl, pH 2.5. + -. 0.1), a CEX mobile phase A (20.0 mmol/L MES, pH 6.5. + -. 0.1), a CEX mobile phase B (20.0 mmol/L MES, 150 mmol/L NaCl, pH 6.5. + -. 0.1) were prepared.

Step (II): cell culture sample preparation

Before the cell culture sample enters the first dimension chromatographic separation device 20, the concentration of the monoclonal antibody in the sample can be estimated by ultraviolet spectrophotometry. According to the measured concentration of the cell culture sample, the cell culture sample is processed by means of dilution or multiple sample introduction and the like and then enters the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30 for analysis.

Step (three): the preparation of the system 100 for on-line monitoring of the yield and quality of a target protein is as follows:

1. before the start of monitoring, the channels involved in the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30 are cleaned with ultrapure water. The corresponding flow phase is then connected to the system and each flow path is flushed in turn. The first detector 23, the second detector 33 are turned on synchronously, and preheated for 30 min.

2. Adjusting the flow speed to 0.2 mL/min, observing the flow directions of the chromatographic columns in the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30, connecting a plurality of chromatographic columns into a column incubator according to the correct flow direction according to the flow direction on the multi-column-position column incubator, connecting a left pump 51 with a Protein A affinity chromatographic column, connecting a right pump 52 with a CEX chromatographic column, washing the chromatographic column for 5 min by using a mobile phase A of the corresponding chromatographic column, and washing the chromatographic column for 5 min by converting into a mobile phase B of the corresponding chromatographic column. Slowly adjusting the flow rate of the left pump 51 to 1.6mL/min, slowly adjusting the flow rate of the right pump 52 to 1.0 mL/min, washing the chromatographic column for 5 min respectively by using the corresponding chromatographic column mobile phase A, changing to the corresponding chromatographic column mobile phase B, washing the chromatographic column for 5 min respectively, finally changing to the corresponding chromatographic column mobile phase A, and washing the chromatographic column for 5 min. And (3) chromatographic column balancing: and balancing the chromatographic column to a base line by using the initial flow rate by using each pump mobile phase A, and injecting the sample stably.

3. Parameter setting of the first dimension chromatography separation apparatus 20 and the second dimension chromatography separation apparatus 30:

the parameters are set according to the nature of the cell culture sample (mab sample), which is exemplified by mab 1 sample.

Protein a affinity chromatography parameters:

a chromatographic column: thermo Scientific (USA) PA ID Sensor card column (2.1X 30mm)

Column temperature: 30 ℃; detector parameters: a detection wavelength of 280 nm (or 220 nm); sample introduction mode: 50 mu L (sample volume can be adjusted according to concentration, full loop sample injection mode or partial sample injection mode)

The left pump 51 elution gradient (which may be modified in conversion to flow rate and column volume) is shown in table 1 below:

TABLE 1

The right pump 52 balances the gradient as shown in table 2 below:

TABLE 2

The first polygonal valve 46 is switched (position-adjustable depending on the flow path connection) as shown in table 3 below:

TABLE 3

The parameters of the collector 312 of the first sample injection module 21 are set as shown in table 4 below:

TABLE 4

Collection position setting of collector 312:

each time a new sequence is run, the collection Tube Position "is set to RA1, and the collection Tube number" is set to 1.

Cation exchange Chromatography (CEX) parameter settings:

a chromatographic column: thermo Scientific (USA) ProPac WCX-10 (4X 250 mm, 10 μm) (the same type of ion exchange chromatography column can be replaced and the corresponding elution gradient needs to be changed)

Left pump 51 elution gradient, table 5 below:

TABLE 5

The right pump 52 elution gradient (for example CEX elution gradient of mab 1, corresponding elution gradients can be used for different mab products) as shown in table 6 below:

TABLE 6

The first polygonal valve 46 is switched (position-adjustable depending on the flow path connection) as shown in table 7 below:

TABLE 7

Step (IV): the control device 10 performs data analysis

The integration events of the CEX spectra were adjusted to ensure that all chromatographic peaks were integrated. Calculating the percentage of peak area of CEX spectrogram:

% acidic component peak area = acidic component peak area/chromatographic peak total peak area × 100%;

% principal component peak area = principal component peak area/chromatographic peak total peak area × 100%;

% basic component peak area = basic component peak area/chromatographic peak total peak area × 100%;

the results of this data analysis are shown in fig. 7, where several characteristic aspects of the analysis:

1. the specificity is as follows: the blank solution has no interference at the peak position in the sample Protein A affinity chromatogram and the CEX chromatogram.

2. Linearity and range: when the sample is purified by the first dimension Protein A affinity chromatography, the recovery rate is not less than 70 percent when the sample injection amount is in the range of 200-350 mu g.

3. Repeatability: when the sample amount is 200 mug, three needles are continuously and repeatedly injected, and the RSD of the CEX main component peak area percentage can reach below 1.0%.

4. Precision: when the sample amount is 200 mug, 250 mug, 300 mug and 350 mug in sequence, the RSD of the CEX main component peak area percentage reaches below 2.5%.

Referring to FIG. 4, in one embodiment of the present invention, the cell culture broth containing the protein of interest is passed through cation exchange chromatography column 31 as described above, and is run at CEX, replacing the analysis method of CEX, and the analysis time is further reduced; before the step C, the following steps (one) to (three) are required to be carried out;

Step (II): cell culture sample preparation

Before the cell culture sample enters the first dimension chromatographic separation device 20, the concentration of the monoclonal antibody in the sample can be estimated by ultraviolet spectrophotometry. According to the concentration of the measured sample, the sample is processed by means of dilution or multiple sample introduction and the like and then enters the first-dimension chromatographic separation device 20 for analysis.

Preparing a standard curve solution from a reference substance corresponding to a monoclonal antibody sample, wherein the diluted solution is Protein A affinity chromatography mobile phase A, and the concentration range is 0.25-4.0 mg/mL.

1. before the start of the detection, the channels involved in the first dimension chromatography separator 20 and the second dimension chromatography separator 30 are cleaned with ultrapure water. The corresponding flow phase is then connected to the system and each flow path is flushed in turn. The first detector 23, the second detector 33 are turned on synchronously, and preheated for 30 min.

2. Adjusting the flow speed to 0.2 mL/min, observing the flow directions of the chromatographic columns in the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30, connecting a plurality of chromatographic columns into a column incubator according to the correct flow direction according to the flow direction on the multi-column-position column incubator, connecting a left pump 51 with a Protein A affinity chromatographic column, connecting a right pump 52 with a CEX chromatographic column, washing the chromatographic column for 5 min by using a mobile phase A of the corresponding chromatographic column, and washing the chromatographic column for 5 min by converting into a mobile phase B of the corresponding chromatographic column. Slowly adjusting the flow rate of the left pump 51 to 1.6mL/min, slowly adjusting the flow rate of the right pump 52 to 0.7 mL/min, washing the chromatographic column for 5 min respectively by using the corresponding chromatographic column mobile phase A, changing to the corresponding chromatographic column mobile phase B, washing the chromatographic column for 5 min respectively, finally changing to the corresponding chromatographic column mobile phase A, and washing the chromatographic column for 5 min. And (3) chromatographic column balancing: and balancing the chromatographic column to a base line by using the initial flow rate by using each pump mobile phase A, and injecting the sample stably.

the parameters are set according to the properties of the monoclonal antibody sample, and the monoclonal antibody 2 sample is taken as an example.

Protein a affinity chromatography parameters:

Column temperature: 30 ℃; detector parameters: a detection wavelength of 280 nm (or 220 nm); sample introduction mode: 100 mu L (sample volume can be adjusted according to concentration, full loop sample injection mode or partial sample injection mode)

The left pump 51 elution gradient (which may be modified in conversion to flow rate and column volume) is shown in table 8 below:

TABLE 8

The right pump 52 balances the gradient as shown in table 9 below:

TABLE 9

The first polygonal valve 46 is switched (position-adjustable according to the flow path connection) as shown in table 10 below:

watch 10

The parameters of the collector 312 of the first sample injection module 21 are set as shown in the following table 11:

TABLE 11

Collection position setting of collector 312:

Cation exchange Chromatography (CEX) parameter settings:

a chromatographic column: thermo Scientific (USA) ProPac Elite WCX (4X 150 mm, 5 μm) (ion exchange chromatography column of the same type can be replaced, the corresponding elution gradient needs to be changed) column temperature: 30 ℃; detector parameters: a detection wavelength of 280 nm (or 220 nm); sample introduction mode: 20 mu L (sample volume can be adjusted according to concentration, full loop sample injection mode or partial sample injection mode)

The left pump 51 elution gradient is shown in table 12 below:

TABLE 12

The right pump 52 elution gradient (for example CEX elution gradient of mab 2, corresponding elution gradients can be used for different mab products) as shown in table 13 below:

watch 13

The switching setting (position adjustment according to the flow path connection) of the first polygonal valve 46 is as shown in table 14 below:

TABLE 14

Step (IV): the control device 10 performs data analysis;

the integration events of the CEX spectra were adjusted to ensure that all chromatographic peaks were integrated. Calculating the percentage of peak area of CEX spectrogram: % acidic component peak area = acidic component peak area/chromatographic peak total peak area × 100%;

referring to FIG. 4, in one embodiment of the present invention, the purity and molecular size heterogeneity of the mAb sample is analyzed by passing the cell culture broth containing the protein of interest through size exclusion chromatography column 32 as described above, followed by SEC instead of SEC. The analysis time is further reduced; before the step S601 is performed, the following steps (a) to (b) need to be performed;

step (I): reagent formulation (exemplified by Protein a affinity chromatography and size exclusion two-dimensional chromatography). The preparation of the reagent can be carried out according to the times described below, and the content of the components of the reagent is ensured to be unchanged.

A hydrochloric acid solution (1.2 mol/L), a sodium hydroxide solution (4.0 mol/L), a Protein A mobile phase A (10.0 mmol/L phosphate, 2.7 mmol/L KCl and 137.0 mmol/L NaCl, pH 7.4), a Protein A mobile phase B (12.0 mmol/L HCl, 150.0 mmol/L NaCl, pH 2.5. + -. 0.1), and a SEC mobile phase A (100 mmol/L phosphate buffer, 150 mmol/L NaCl, pH 6.8. + -. 0.2) were prepared.

Step (II): cell culture sample preparation

Before the cell culture sample enters the first dimension chromatographic separation device 20, the concentration of the monoclonal antibody in the sample can be estimated by ultraviolet spectrophotometry. According to the concentration of the measured sample, the sample is processed by means of dilution or multiple sample introduction and the like and then enters the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30 for analysis. Preparing a standard curve solution from a reference substance corresponding to a monoclonal antibody sample, wherein the diluted solution is Protein A affinity chromatography mobile phase A, and the concentration range is 0.25-4.0 mg/mL.

2. Adjusting the flow speed to 0.2 mL/min, observing the flow directions of the chromatographic columns in the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30, connecting a plurality of chromatographic columns into a column incubator according to the correct flow direction according to the flow direction on the multi-column-position column incubator, connecting a left pump 51 with a Protein A affinity chromatographic column, connecting a right pump 52 with an SEC chromatographic column, washing the chromatographic column for 5 min by using a corresponding chromatographic column mobile phase A, and washing the chromatographic column for 5 min by converting into a chromatographic column corresponding mobile phase B. Slowly adjusting the flow rate of the left pump 51 to 1.6mL/min, slowly adjusting the flow rate of the right pump 52 to 0.5 mL/min, washing the chromatographic column for 5 min respectively by using the corresponding chromatographic column mobile phase A, changing to the corresponding chromatographic column mobile phase B to wash the chromatographic column for 5 min respectively, finally changing to the corresponding chromatographic column mobile phase A, and washing the chromatographic column for at least 30 min. And (3) chromatographic column balancing: and balancing the chromatographic column to a base line by using the initial flow rate by using each pump mobile phase A, and injecting the sample stably.

the parameters are set according to the nature of the cell culture sample (mab sample), which is exemplified by mab 2 sample.

Protein a affinity chromatography parameters:

The left pump 51 elution gradient (which can be modified in conversion to flow rate and column volume) is shown in table 15 below:

watch 15

The right pump 52 balances the gradient as in table 16 below:

TABLE 16

The first polygonal valve 46 is switched (position adjustment is possible depending on the flow path connection) as shown in table 17 below:

TABLE 17

The parameters of the collector 312 of the first sample injection module 21 are set as shown in the following table 18:

watch 18

Collection position setting of collector 312:

Size Exclusion Chromatography (SEC) parameter settings:

a chromatographic column: waters (USA) Xbridge BEH SEC (7.8X 300 mm, 3.5 μm) (size exclusion chromatography column 32 of the same type and size can be replaced and the corresponding elution gradient needs to be changed)

The left pump 51 elution gradient is shown in table 19 below:

watch 19

The right pump 52 elution gradient (SEC elution gradient for mab 2 as an example) is shown in table 20 below:

watch 20

The first polygonal valve 46 is switched (position-adjustable depending on the flow path connection) as shown in table 21 below:

TABLE 21

Step (IV): the control device 10 performs data analysis, see fig. 8;

the integration events of the SEC spectra were adjusted to ensure that all chromatographic peaks were integrated. Peak area percentage calculation of SEC spectra:

% area of high polymer peak = area of high polymer peak/total area of chromatographic peak × 100%;

% monomer peak area = monomer peak area/total peak area of chromatographic peak × 100%;

% low molecular weight substance peak area = low molecular weight substance peak area/chromatographic peak total peak area × 100%.

Referring to fig. 1, 2 and 5, in one embodiment of the invention, the second dimension chromatographic separation device 30 comprises a cation exchange chromatography column 31 and a size exclusion chromatography column 32; the Protein A affinity chromatographic column 22 and the cation exchange chromatographic column 31 are connected with the first detector 23 through a third multi-angle valve 48; or the Protein A affinity chromatographic column 22 and the size exclusion chromatographic column 32 are connected with the first detector 23 through a third multi-angle valve 48;

a cation exchange chromatography column 31 or a size exclusion chromatography column 32 is connected to the second detector 33;

the control device 10 further includes: a parallel path selection module 19, a fifth control module 17, and a second calculation module 18;

the step S602 includes:

b5, the parallel path selection module 19 sets the eluent containing the target protein through the cation exchange chromatography column 31 and the size exclusion chromatography column 32 by controlling the path of the third multi-angle valve 48;

b6, the cation exchange chromatographic column 31 and the size exclusion chromatographic column 32 respectively perform cation exchange chromatographic separation and size exclusion chromatographic separation on the eluent containing the target protein to obtain a target protein solution;

b7, the fifth control module 17 sends a sixth control signal to the second detector 33 and the first detector 23, detects the ultraviolet absorption signal of the eluate containing the target protein passing through the cation exchange chromatography column 31 and the size exclusion chromatography column 32, and returns the second detection signal to the control device 10;

b8, the second calculating module 18 calculates the charge heterogeneity, monomer purity and molecular size heterogeneity of the target protein solution according to the second detection signal.

In this example, the two-dimensional liquid phase formed by the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30 is connected with Protein A affinity chromatography, SEC and CEX chromatographic columns in parallel, and the yield of the target Protein solution, SEC and CEX results are obtained by simultaneous analysis. The efficiency and accuracy of on-line monitoring of yield and quality of mab upstream fermentation broth will be higher due to the simultaneous results obtained for SEC and CEX. After the cell culture sample containing the target Protein is taken out from the culture tank, the cell culture sample flows into a sampling ring 311 of a sampling system in a two-dimensional liquid phase sampling system after centrifugal filtration, the Protein of the cell culture fermentation liquid containing the target Protein is captured onto a chromatographic column by the first dimension of Protein A affinity chromatography, and the Protein is eluted from the chromatographic column by adopting an elution phase through the setting of the control device 10 to obtain an elution solution containing the target Protein. The control device 10 may be implemented as a computer control system. Then, the target protein solution flows through the first detector 23, the control device 10 calculates the yield information of the target protein solution containing the target protein eluent, the information is fed back to the control device 10, and the control device 10 controls the sample to be collected into the collector 312 of the sample injection system; the first sample injection module 21 can take out the sample separated and collected by the first dimension chromatography, that is, the eluent containing the target protein is pumped into the second dimension chromatography and sequentially pumped into the CEX and SEC chromatography columns connected in parallel in the multi-column-position column incubator 45, and then respectively flows through the corresponding first detector 23 and second detector 33 (wherein one detector is shared by the first dimension chromatography and the parallel flow paths) through the separation of the cation exchange chromatography column 31 and the size exclusion chromatography column 32, and the control module 10 simultaneously calculates the information of the charge heterogeneity, the monomer purity and the molecular size heterogeneity of the protein according to the feedback information.

In one embodiment of the invention, the cation exchange column 31 and the size exclusion column 32 of the previous embodiment are operated in parallel flow paths. In the case of a serial flow arrangement of cation exchange column 31 and size exclusion column 32, the two-dimensional liquid autosampler module has a sampling loop for collecting the stored sample. In the present embodiment, the capture and elution of the sample in the Protein a affinity chromatography column are achieved by the switching of a multi-angle valve, such as a six-way valve. The eluted monoclonal antibody sample can be temporarily stored in a sample injection vial in an automatic sample injector, a sample injection needle is operated through program setting, the sample is sequentially sent into parallel CEX and SEC chromatographic columns, and finally the charge heterogeneity, purity and molecular size heterogeneity information of the monoclonal antibody product is obtained simultaneously.

1, capturing a cell culture sample. The six-way valve is connected as shown in FIG. 9, the sample in the flow path flows through 2_1, passes through the Protein A affinity chromatographic column, the Protein is trapped in the chromatographic column, and the residual liquid flows into the waste liquid from the flow path of 4_ 3; at this time, the elution phase of Protein A (Protein A mobile phase B) passed through the flow channel 6_ 5.

And 2, eluting the cell culture sample. The six-way valve is connected as shown in fig. 10, and a sample in the flow path flows through 2_3 and is directly transferred into waste liquid or in a state of stopping sampling; the elution phase of Protein a (Protein a mobile phase B) will be connected to the Protein a column from the flow path 6_1, and the captured Protein will be eluted from the column, and then flow through 4_5 into the collector 312 of the sample injection system.

In the above embodiments, the cell culture sample is first passed through the Protein a affinity chromatography system of the first dimension chromatography separation apparatus 20 to obtain the concentration (titer) of the sample; then, through central cutting, antibody peaks in Protein A affinity chromatography are collected and enter a second-dimensional cation exchange or size exclusion chromatography of a second-dimensional chromatographic separation device 30, and the charge heterogeneity or purity and molecular size heterogeneity of a sample is analyzed; the whole process is automatically completed by the control device 10 according to the set program without manual intervention. Can be directly connected to cell culture equipment to realize the purpose of on-line monitoring. Besides monitoring the yield attribute (titer) of the monoclonal antibody product, the method can also realize the preliminary purification and enrichment of the sample, and simultaneously monitor the charge heterogeneity (CEX), purity and molecular size heterogeneity (SEC) information of the product. In an embodiment of the present invention, after 2.0 to 2.5 minutes of the sample in the Protein a affinity chromatography chromatogram is collected, the collected sample and the second-dimensional chromatography mobile phase are mixed at a certain ratio (e.g., 2: 1, 1.5: 1, etc.) by setting the apparatus and switching the valves. The process of mixing the collected sample in the first dimension and the mobile phase in the second dimension can be realized by various means, such as a sample loop 311, a sample bottle, etc. Through the specific method setting, the volume of the second-dimension sample injection can be adjusted according to the yield information (titer) obtained by the first dimension, so as to ensure that the protein content of the second-dimension sample injection meets the requirement. Aiming at a monoclonal antibody product with low concentration (less than 2.0 mg/mL), the signal intensity and the separation efficiency are improved by repeating the sample injection mode of the first dimension Protein A affinity chromatography. Moreover, through the above experimental examples, it can be seen that the results of analyzing the yield and quality attributes of the monoclonal antibody product simultaneously by two-dimensional liquid phase should be consistent with the results of analyzing the yield and quality by the same chromatographic method, respectively, and the Relative Standard Deviation (RSD) is less than or equal to 4.0%. The yield and quality attributes of the monoclonal antibody product are analyzed simultaneously through the two-dimensional liquid phase, the whole analysis time can be reduced from the traditional 4 hours/sample to less than 1 hour/sample, and the monitoring efficiency is greatly improved.

As can be seen from the above embodiments, the system 100 provided by the present invention can realize the automation and continuity of online monitoring of yield and quality (charge heterogeneity or purity and molecular size heterogeneity) in the monitoring process using the system 100, automatically complete the sample pre-processing step from manual transfer to an instrument, shorten the analysis completion time, reduce the operation steps, reduce the error probability, and can perform specific development and verification on the product process.

In addition, the method provided by the invention further comprises the following steps:

the first pump module 50 provides motive force for mobile phase, isocratic or gradient elution when the first dimension chromatographic separation device 20 and the second dimension chromatographic separation device 30 carry out chromatographic separation; and/or

The second dimension chromatographic separation device 30 conveys the waste liquid generated by the separation thereof to the waste liquid recoverer 60.

In summary, the present invention provides a method for online monitoring of yield and quality of a target protein, which is implemented by using a system for online monitoring of yield and quality of a target protein, the system for online monitoring of yield and quality of a target protein includes a control device, a first dimension chromatography separation device and a second dimension chromatography separation device, wherein the first dimension chromatography separation device and the second dimension chromatography separation device form a two-dimensional liquid phase system, and thus, after being connected to a cell culture sampling device for real-time sampling in a cell culture process, the two-dimensional liquid phase system is directly connected to a two-dimensional liquid phase for analysis of yield and quality (charge heterogeneity or purity and molecular size heterogeneity) after online centrifugation or filtration: therefore, the yield and the quality of the upstream fermentation liquor of the target protein solution can be automatically monitored, and the monitoring efficiency is improved.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it should be understood that various changes and modifications can be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for on-line monitoring of the yield and quality of a target protein, wherein the method is implemented by using a system for on-line monitoring of the yield and quality of a target protein; the system comprises: the device comprises a control device, a first dimension chromatographic separation device and a second dimension chromatographic separation device; the method comprises the following steps:

A. the first dimension chromatographic separation device receives a control signal of the control device, performs protein A affinity chromatographic separation on the cell culture fermentation broth containing the target protein to obtain an eluent containing the target protein, and returns a first detection signal to the control device; the control device calculates the yield information of the target protein solution according to the first detection signal;

2. The method of claim 1, wherein the system further comprises: a cell culture sampling device; the method further comprises the following steps:

3. The method of claim 1, wherein the first dimension chromatographic separation device comprises a first sample injection module, a Protein A affinity chromatographic column and a first detector;

the A comprises the following steps:

4. The method of claim 3, wherein the second dimension chromatographic separation device comprises a cation exchange chromatography column, a size exclusion chromatography column, and a second detector; the Protein A affinity chromatographic column, the cation exchange chromatographic column and the size exclusion chromatographic column are connected with the collector through a second multi-angle valve;

the B comprises:

5. The method of claim 3, wherein the second dimension chromatographic separation device comprises a cation exchange chromatographic column and a size exclusion chromatographic column; the Protein A affinity chromatography column, the cation exchange chromatography column, or the Protein A affinity chromatography column and the size exclusion chromatography column are connected with the first detector through a third multi-angle valve;

the B comprises:

6. The method of claim 2, wherein the cell culture sampling device comprises a cell culture tank, a centrifuge and/or a filter connected to each other; the control device further includes: a fourth control module;

the C comprises:

7. The method of claim 1, wherein the system further comprises: the first pump module is connected with the first sample introduction module and the second dimensional chromatographic separation device, and the waste liquid recoverer is connected with the second dimensional chromatographic separation device;

the method further comprises the following steps:

8. The method of claim 4 or 5, wherein the Protein A affinity, cation exchange, and size exclusion chromatography columns are arranged in a multi-column incubator;

the first detector and the second detector are both ultraviolet detectors;

the system also includes a first pump module comprising a left pump and a right pump; the left pump is connected with the Protein A affinity chromatographic column, and the right pump is connected with the cation exchange chromatographic column and/or the size exclusion chromatographic column;

the multi-angle valve is a hexagonal valve.

9. The method of claim 1, wherein the cell culture sample is a cell culture sample comprising a protein of interest.