WO2021197248A1 - 连续纯化生物制药产品的系统和方法 - Google Patents

连续纯化生物制药产品的系统和方法 Download PDF

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WO2021197248A1
WO2021197248A1 PCT/CN2021/083516 CN2021083516W WO2021197248A1 WO 2021197248 A1 WO2021197248 A1 WO 2021197248A1 CN 2021083516 W CN2021083516 W CN 2021083516W WO 2021197248 A1 WO2021197248 A1 WO 2021197248A1
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main unit
chromatography
buffer volume
concentration
unit
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PCT/CN2021/083516
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English (en)
French (fr)
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巩威
陈然
姚彬
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上海复宏汉霖生物技术股份有限公司
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Priority claimed from CN202020432845.9U external-priority patent/CN212379272U/zh
Priority claimed from CN202011400115.1A external-priority patent/CN114573689A/zh
Application filed by 上海复宏汉霖生物技术股份有限公司 filed Critical 上海复宏汉霖生物技术股份有限公司
Publication of WO2021197248A1 publication Critical patent/WO2021197248A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the invention relates to the field of biopharmaceuticals, and in particular to a continuous flow purification process and a system for purifying biopharmaceutical products.
  • the target molecules of biopharmaceuticals are usually macromolecules, and the production process is relatively complicated, including multiple purification steps and strict cleaning and sterilization and virus removal processes.
  • the complexity of the production of biological products and in order to meet the technical requirements and regulatory specifications for the production of pharmaceutical products, it is necessary to detect and control multiple indicators during the production process, which is not conducive to the continuous purification of pharmaceutical production, resulting in poor production efficiency. High, insufficient production capacity, and expensive products.
  • WO2020148119A1 discloses a method for converting a batch purification process of monoclonal antibodies into a continuous purification process.
  • the downstream process of antibody uses protein A (Protein A) affinity chromatography, cation exchange chromatography, anion exchange chromatography, virus filtration and ultrafiltration/dialysis.
  • Protein A affinity chromatography uses Bio SMB chromatography with 5 chromatographic columns, which can generate a continuous feed stream and a continuous eluent stream.
  • CEX adopts parallel batch mode, and both virus filtration and ultrafiltration/dialysis adopt batch mode.
  • CN106794424A discloses a control system and method used by a connection system in fluid communication with a tangential flow filtration processing unit, such as a chromatography processing unit.
  • the present invention provides a purification system for continuous purification, which is used for continuous, closed and automated purification of target molecules.
  • the present invention provides a purification system for continuous purification, which comprises
  • each affinity chromatography main unit independently contains one or more affinity chromatography subunits
  • At least one virus inactivation main unit wherein the virus inactivation main units each independently comprise one or more virus inactivation subunits;
  • each concentration and/or liquid exchange main unit independently contains one or more concentration and/or liquid exchange sub-units
  • One or more buffer volumes optionally present independently upstream or downstream of each main unit; each of the main units and the buffer volume is connected by a pipeline that allows the flow of material and liquid,
  • each buffer volume is configured with at least one weight sensor and/or at least one liquid level sensor, and the weight sensor and the liquid level sensor are in communication connection with the management system;
  • the management system controls the starting, continuing and stopping of the purification system according to the signal of the weight sensor and/or the liquid level sensor;
  • the target molecule purified by the purification system is a protein
  • the concentration and/or liquid exchange unit is a unit for implementing ultrafiltration or dialysis performed in a continuous operation.
  • the flow direction of the stream containing the sample is different from the filtration direction .
  • the present invention provides a method of purifying target molecules, which is accomplished by the system of the present invention.
  • Figure 1 is a schematic diagram of an operation layer including a convergence layer.
  • Figure 2 is a schematic diagram of the monoclonal antibody purification system.
  • Figure 3 is a schematic diagram of the online target protein quantitative detection system.
  • Figure 4 is a schematic diagram of the online target protein quantitative detection system.
  • Figure 5 shows the connection between the flow cell in the online target protein quantitative detection system and other devices of the system.
  • Fig. 6 is a schematic diagram of the structure of the flow cell in the online target protein quantitative detection system.
  • Fig. 7 is an enlarged view of A in Fig. 6.
  • Figure 8 is a schematic diagram of an online HPLC device.
  • Figure 9 is a schematic diagram of a purification system with a convergent layer (the main unit of cation exchange chromatography serves as the convergent layer).
  • Figure 10 is a schematic diagram of a purification system with two convergent layers (the main unit of cation exchange chromatography serves as the convergent layer).
  • metering ratio refers to the proportioning of various substances according to a certain weight.
  • processing step or “unit operation” used interchangeably herein refers to the use of one or more methods or devices in the purification process to achieve a certain result.
  • processing steps or unit operations that can be used in the purification process include, but are not limited to, clarification, chromatography, virus inactivation, and concentration and/or liquid exchange. It should be understood that each processing step or unit operation can adopt more than one step or method or device to achieve the expected result of the processing step or unit operation.
  • material conversion is broadly defined as the conversion of a specific material into another material with different characteristics under the influence of physical, chemical or biological conditions. This conversion can be embodied as changes in physical properties (such as melting, dissolution, etc.), changes in chemical properties (such as chemical reactions, such reactions can be carried out under biological conditions, such as enzyme-catalyzed reactions) or as a mixture of materials in Changes in composition (e.g. purification process).
  • materials refer to the objects of production and processing, as well as the products that are produced and processed. Generally speaking, “material” refers to the material objects that the production process acts on, and it covers the time span from the beginning of the production process (raw material) to the end of the production process (final product).
  • batch production or “batch manufacturing” mean the same meaning and can be used interchangeably herein.
  • Batch production is characterized by “batch process” (also called “batch process”, “batch process”) or “batch operation” Production.
  • continuous production or “continuous manufacturing” or “continuous purification” (continuous manufacturing, CM, or continuous purification) refers to a continuous process (also called “continuous process”) or continuous operation (Continuous operation) is the characteristic production or purification.
  • continuous refers to the continuous input of raw materials into the reactor, and the continuous output of reaction products from the reactor. At each point in time, there is a movement of materials that are input as raw materials and output as products.
  • Continuous production or continuous manufacturing” or “continuous purification” realizes continuous, closed, and automated separation and purification of target molecules, including at least two CM unit operations that overlap in time at least partially.
  • Continuous manufacturing can improve pharmaceutical manufacturing in, for example, the following aspects.
  • the use of integrated processes reduces the number of steps and shortens the processing time; the required equipment area is small; supports enhanced development methods (for example, design quality (QbD) and the use of process analysis techniques, and the use of mathematical models); real-time monitoring of product quality ; And provide flexible operation to make it easy to scale-up, scale-down and scale-out to adapt to changing supply requirements. Therefore, the use of continuous manufacturing for drug production is expected to reduce drug quality problems, reduce manufacturing costs, and improve patients' chances of obtaining high-quality drugs.
  • Continuous manufacturing in the "bin to bin” mode refers to the continuous material transfer between single, disconnected CM unit operations, which is achieved at a higher level of granularity in the process design Continuous operation of the process chain.
  • the continuous manufacturing method of the present invention refers to a method of purifying target molecules, which includes two or more processing steps (or unit operations), so that the output from one processing step flows directly into the next processing step in the method without interruption and /Or there is no need to collect the entire volume of output from the processing step before proceeding to the next processing step.
  • two or more processing steps can be performed simultaneously for at least a portion of their duration.
  • continuous is also used for steps within a processing operation, in which case, during the processing operation that includes multiple steps, the sample continuously flows through the multiple steps required to perform the processing operation.
  • An example of such a processing operation described herein is an affinity chromatography step and a concentration and/or liquid exchange step.
  • Such a processing operation described herein is a polishing purification operation, which includes multiple steps performed in a continuous manner.
  • processing operations include two or more of CEX chromatography, anion exchange/cation exchange mixed media treatment, and AEX chromatography.
  • concentration and/or liquid exchange steps described herein refer to reducing the volume of liquid in the fluid material and/or exchanging the buffer system.
  • the specific function of this step is concentration, liquid exchange, or concentration and liquid exchange, which can be judged by those skilled in the art according to the characteristics of the step itself and/or the characteristics of the steps adjacent to the concentration and/or liquid exchange step in the process flow.
  • methods that can be adopted for the concentration and/or fluid exchange step include filtration and dialysis. For filtration, for example, a membrane method (for example, ultrafiltration) or gel filtration chromatography can be used.
  • the filtration can be tangential flow filtration (cross flow filtration) or dead-end filtration (normal flow filtration or dead-end filtration), preferably tangential flow filtration, especially unidirectional tangential flow filtration (single-flow filtration). pass tangential flow filtration, SPTFF).
  • the dialysis is preferably in-line diafiltration (ILDF).
  • continuous methods also include methods in which the input or output of fluid substances in any single unit operation is discontinuous or intermittent. Such a method can also be referred to as a "semi-continuous" method.
  • input in unit operations e.g., binding and elution chromatography steps
  • output can be collected intermittently, where other unit operations in the purification process are continuous. Therefore, in some embodiments, the methods and systems described herein include at least one unit operation performed as a batch operation, while other unit operations in the method or system can be operated in a continuous manner.
  • the function of the "buffer volume” in this article is to realize the adjustment of the material distribution in a continuous process, and to provide a buffer to allow the material to flow at a desired flow rate between unit operations.
  • the buffer volume can be in any known form, such as a container (e.g. a buffer tank, a reservoir bag), or an expandable tube. It should be understood that the container may have a completely fixed shape, or may be deformable in whole or in part.
  • buffer tank refers to any container or vessel or bag used between or within processing steps (for example, when a single processing operation includes more than one step); wherein the output from one step flows through the buffer tank to Next step. Therefore, the buffer tank is different from the collection tank in that it is not intended to hold or collect the entire volume of output from the step; instead, it allows the output from one step to flow continuously to the next step.
  • unit refers to a device or a part of a device used to perform process technical steps, or its operation cycle in units of cycles.
  • the unit can achieve specific functions in the purification system.
  • "Unit” includes but is not limited to “main unit” and “subunit”.
  • "Main unit” refers to a device or a collection of device parts that implements a specific function in a specific step, and can also be understood as a module. Examples of the main unit include, but are not limited to, a chromatography main unit, a concentration and/or liquid exchange main unit, a virus removal main unit, and the like.
  • the main units are connected by a flow path, so that the material undergoes corresponding process steps in the process of passing through each main unit.
  • each main unit runs simultaneously at a similar feed rate in order to minimize production time.
  • the "main unit” is composed of one or more “subunits” that implement the same functions.
  • “Sub-unit” refers to a cycle of operation of a device or part of a device that performs a process technology step, and can also be expressed as a cycle of operation.
  • any affinity chromatography main unit may include one or more affinity chromatography subunits, that is, one or more affinity chromatography columns each run independently for one or more cycles.
  • any cation exchange chromatography main unit, anion exchange chromatography main unit, or virus removal main unit may contain one or more subunits.
  • a chromatography unit may be implemented by any known chromatography device, such as membrane chromatography device, chromatography column, microbeads, magnetic beads and other stationary phase carriers, multi-column flow system, fluid bed chromatography system and other layers. Analysis system, etc.
  • a chromatography subunit refers to an operation cycle of a chromatography column or a membrane chromatography device, especially an operation cycle of a chromatography column.
  • a unit has one or more elements selected from process technology elements, service technology elements, and elements for data collection and processing.
  • the process technology unit includes elements used to perform steps, including but not limited to hoses, filters, chromatography columns, containers, etc., which are not connected to the management system.
  • the service technology unit includes all sensors and actuators of the unit. They are connected to the management system through RIO.
  • the actuator of the STU can be, for example, a pump, a valve, and the sensor can be, for example, a UV measurement, a pressure sensor, or a weight sensor.
  • the components used for data acquisition and processing are remote I/O or local intelligence, such as programmable logic control (PLC) or PC-based systems with an I/O layer. Perform basic automation of the unit on local control.
  • PLC programmable logic control
  • RIO registered input/output
  • substream refers to the stream formed by the output of material from a certain unit to its adjacent downstream unit.
  • a sub-stream refers to the material stream formed by a certain sub-unit to its neighboring downstream unit continuously in time for the primary output of materials.
  • the affinity chromatography main unit is a multi-column continuous affinity chromatography device
  • one operation cycle of any chromatography column is an affinity chromatography subunit
  • the output of each affinity chromatography subunit The stream serves as an input stream for the next step (adjacent downstream unit).
  • the output stream from the affinity chromatography main unit to the adjacent downstream unit includes multiple substreams.
  • the output streams of two sub-units are continuous with each other, which means that when the output stream of the first sub-unit ends, the output stream of the second sub-unit just starts, that is, the output streams of the two sub-units are in time. It is continuous, and it can be considered that the two output streams together form a continuous stream.
  • the situation where the output streams of two or more subunits are continuous with each other should also be understood similarly.
  • the operation time of two subunits is continuous with each other means that when the operation of the first subunit ends, the operation of the second subunit just starts, that is, the operations of the two subunits are continuous in time. .
  • a running cycle corresponds to the process of a certain amount of sample input and sample output, and the time of sample input and sample output can be partially overlapped, completely overlapped, or completely non-overlapped.
  • chromatography is performed in a binding-elution mode, where one operating cycle includes a process (ie, a certain amount) of sample input (binding)-sample output (elution).
  • one operating cycle of a chromatography column The time of sample input and sample output partially overlaps or does not overlap at all, and preferably does not overlap at all.
  • the time of sample input and the time of sample output are respectively completely continuous.
  • a specific length of time can be designated as a running cycle, and the specific length of time corresponds to The specific sample input volume and/or specific sample output volume, and the specific sample input volume and/or specific sample output volume correspond to the product volume output by the immediately upstream unit during one operation cycle. That is to say, when dividing the operating cycle of such a device or part of the device, the time that the device or part of the device is used to process samples generated in one operating cycle of the immediately upstream unit and output downstream as a product can be divided into one according to needs. Operation cycle.
  • the time for processing the samples generated in one operating cycle of the immediately upstream unit includes the time of receiving the sample (sample loading), so the start time of the operating cycle is the time to start receiving the sample generated in one operating cycle of the immediately upstream unit.
  • one operation cycle of the main unit can be defined as a sample used to process the samples generated by the operation cycle of the immediately upstream main unit and output downstream as a product. time.
  • the end point of a period of time (for example, one operating cycle) and the starting point of another period of time (for example, another operating cycle) are at the same time point. Such a situation does not belong to the overlap of time periods, and such a situation is also included in the In the case of "no overlap at all".
  • two or more operating cycles may be partially overlapped, completely overlapped or not overlapped at all, and preferably partially overlapped or not overlapped at all.
  • the time for processing samples generated in one operating cycle of the immediately upstream unit in a specific operating cycle of a chromatography column that performs chromatography in flow-through mode can be compared with another operating cycle (e.g., upper
  • the time for outputting products downstream in one operation cycle partially overlaps or completely overlaps, and the degree of overlap can be set in advance according to the need to achieve continuous production.
  • the sample input time and/or sample output time of multiple subunits of a specific main unit can be coordinated as needed to achieve continuous sample input time and/or sample output time of the main unit, or,
  • the buffer volume can also be used as needed to realize the continuous sample output time of the buffer volume immediately upstream of the main unit and/or the continuous buffer volume sample input time of the immediately downstream main unit.
  • any device or device part can optionally run for one or more operating cycles according to product requirements, or according to the amount of sample injected, or according to the length of time the purification system runs, which can be specifically controlled by the management system.
  • the length of a specific operating cycle of any device or device part (for example, any element in the device) that performs a process technology step can be measured, anticipated, and controlled.
  • Traditional antibody chromatography purification steps include: (i) concentration of the target product and removal (capture) of harmful impurities, (ii) optionally, further removal of a large amount of impurities (enhance), (iii) finally, removal of remaining traces Impurities and unwanted structural variants of the target product, such as dimers and multimers (polish, "polish") of the target product.
  • the discussion of the operation of the antibody chromatography purification unit in this article mainly involves affinity chromatography and ion exchange chromatography, and does not exclude the use of any known chromatography methods that can be used to purify antibodies, such as mixed mode chromatography, hydrophobic interaction layer Analysis, size exclusion chromatography, hydroxyapatite chromatography, and any combination of the above-mentioned various chromatography.
  • the chromatography step is selected from affinity chromatography, ion exchange chromatography and combinations thereof.
  • the purification of antibodies also includes the operation of the virus removal unit.
  • the virus removal unit operation can use any known method as long as it meets the product quality requirements of the unit operation set by the process of the present invention. Examples of virus removal unit operations include, but are not limited to, virus inactivation, virus removal filtration, and combinations thereof.
  • process characterization refers to the process of characterizing the process. It includes measuring, categorizing and evaluating parameters.
  • process design space refers to the design space defined by the ICH Q8 guidelines.
  • Design space refers to the multi-dimensional combination and interaction of input variables (for example, material properties) and process parameters that have been proven to provide quality assurance. Therefore, the design space is defined by the important and critical process parameters and their acceptable ranges determined from the process characterization study. The definition itself cannot be extended by the process designer, but needs to be elaborated by the industry and regulatory agencies. The design space of a certain product must undergo regulatory evaluation and approval.
  • the method of establishing design space involves process characterization research, and generally involves three key steps: 1. Conduct risk analysis to identify parameters for process characterization; 2. Use DoE (design of experiments) to design multivariate studies Plan, so that the research can obtain data suitable for understanding and defining the design space; 3.
  • the impact is evaluated based on the process parameters that have a statistically significant impact on CQA, and the process parameters that are determined to have a significant impact on CQA are classified as CPP, and the acceptable range of important and critical process parameters is calculated, for example, through the estimated failure
  • the uncertainty of the edge is analyzed to determine the acceptable range of the parameters.
  • These acceptable ranges together define the design space.
  • the design space can be enlarged/reduced by zooming in/out the mathematical model.
  • DoE is generally used to find the range of instrument operating parameters to understand changes in sample preparation and method accuracy.
  • characterization range refers to the range inspected during process characterization.
  • Metal operable design range refers to the parameters in a method design space that can be changed within the scope allowed by supervision.
  • QbD Quality by Design
  • PAT Process Analytical Technologies
  • the desired goal of the PAT framework is to design and develop a fully understood process, and to always ensure the preset quality at the end of the production process.
  • the process is considered to be fully understood: 1Identify and explain all key sources of change; 2Changes can be managed through the process; 3Product quality attributes can be established by the raw materials used, process parameters, production, environment and other conditions Design the space for accurate and reliable predictions.
  • the application of PAT can obtain a more fundamental understanding of the process, so it has a fundamental improvement compared with traditional biological manufacturing.
  • the application of PAT can help obtain univariate or multivariate statistical process control (SPC or MSPC) models. It should be understood that PAT cannot be achieved only by improving analysis technology.
  • CQA Crohn's Quality Attribute
  • critical process parameter refers to a process parameter that significantly affects CQA in the process.
  • OOS inspection result deviation
  • OOS is a result deviation, and is often caused by production operations.
  • OOS processing generally includes performing corresponding deviation analysis (for example, whether it is due to an error in the experiment, such as: the experiment does not meet the system adaptability or the experiment Acceptance standards (or parts that do not meet the experimental acceptance standards), whether there are abnormalities in technology/instruments, raw materials) and deviation handling procedures.
  • In-line detection is also called in-situ detection.
  • On-line detection technology usually requires modification of the bioreactor to shift the sample stream.
  • Off-line or at-line detection is a discontinuous analysis method, involving sampling or sample pre-processing.
  • Upstream refers to the front or upstream position of the material circulation process in the purification system; “downstream” refers to the downstream or downstream position of the material circulation process in the purification system, and the material circulation process in the entire purification system middle. Whether a certain main unit or buffer volume is located upstream or downstream is relative. Take the virus inactivation main unit as an example. In some embodiments, the affinity chromatography main unit is located upstream of the virus inactivation main unit. The main unit for concentration and/or liquid exchange is located downstream of the main unit for virus inactivation.
  • the "nearby unit” of a unit means that there is no other functional unit existing between a unit and its adjacent unit except for connecting pipes.
  • the adjacent unit can be the main unit or the buffer volume;
  • the "nearby unit” of a certain main unit "Next to the main unit” means that there is no other main unit existing between a main unit and its neighboring main unit except for the connecting pipes and buffer volume;
  • Weight sensor refers to a sensor that can directly or indirectly sense the weight of the "buffer volume” or “buffer tank”;
  • liquid level sensor refers to the height of the liquid level in the “buffer volume” or “buffer tank” directly or indirectly Or location sensor.
  • the present invention provides a purification system for continuous purification, which comprises:
  • each affinity chromatography main unit independently contains one or more affinity chromatography subunits
  • At least one virus inactivation main unit wherein the virus inactivation main units each independently comprise one or more virus inactivation subunits;
  • each concentration and/or liquid exchange main unit independently contains one or more concentration and/or liquid exchange sub-units
  • One or more buffer volumes optionally present in the upstream or downstream of each main unit; each of the main units and the buffer volume is connected by a pipeline that allows the material and liquid to circulate,
  • each buffer volume is configured with at least one weight sensor and/or at least one liquid level sensor, and the weight sensor and the liquid level sensor are in communication connection with the management system;
  • the management system controls the starting, continuing and stopping of the purification system according to the signal of the weight sensor and/or the liquid level sensor;
  • the target molecule purified by the purification system is a protein
  • the concentration and/or main liquid exchange unit is a unit used to implement ultrafiltration or dialysis in a continuous operation.
  • the flow direction of the stream containing the sample is related to the filtration The direction is different.
  • the target molecule purified by the purification system is a protein, preferably an antibody, especially a monoclonal antibody.
  • the main unit of affinity chromatography is selected from protein A affinity chromatography, protein G affinity chromatography, and protein L affinity chromatography according to the properties of the target molecule.
  • the target molecule purified by the purification system is a monoclonal antibody containing an Fc fragment.
  • the target molecule purified by the purification system is a fusion protein containing an Fc fragment.
  • the management system controls the following operations based on the weight and liquid level signals:
  • the purification system starts to run.
  • the purification system is started.
  • the main unit When the feedback value of the weight sensor and/or the feedback value of the liquid level sensor of the upstream buffer volume of any main unit reaches the preset value, start the operation of the main unit. In a preferred embodiment, when the feedback value of the weight sensor of the buffer volume upstream of any main unit reaches a preset value, the main unit is started to run. In another preferred embodiment, when the feedback values of the weight sensor and the liquid level sensor of the buffer volume upstream of any main unit both reach the preset value, the main unit is started to run.
  • the present invention also provides a purification system for continuous purification as described above, which in turn includes at least one affinity chromatography main unit, at least one virus inactivation main unit, and at least one concentration and/or Exchange the main unit; and after at least one affinity chromatography subunit target liquid of at least one affinity chromatography main unit flows out of the affinity chromatography main unit, the management system allows at least one of the viruses At least one virus inactivation subunit of the inactivation main unit starts operation; after the target liquid of at least one virus inactivation subunit of the at least one virus inactivation main unit flows out of the virus inactivation main unit, the management system At least one concentration and/or liquid exchange subunit of at least one of the concentration and/or liquid exchange main unit is allowed to start up operation, and the management system allows the target liquid of the first subunit of the affinity chromatography main unit to flow out After the affinity chromatography main unit, the running time of the virus inactivation main unit and the affinity chromatography main unit at least partially overlap; the management system allows the
  • the running time of more than one unit overlaps at least partially, it means that the running time of the units overlaps on the time axis, and it does not limit the running of the one or more units to start at the same time point. Or end at the same point in time.
  • the expression “at least a part of the operating time overlaps” means that about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, About 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95 % Or about 100% overlap.
  • the present invention also provides a purification system for continuous purification as described above, further comprising at least one cation exchange layer between the affinity chromatography main unit and the concentration and/or liquid exchange main unit.
  • Analysis main unit and/or at least one anion exchange chromatography main unit said cation exchange chromatography main unit each independently comprising one or more cation exchange chromatography subunits, said anion exchange chromatography main unit each independently Contains one or more anion exchange chromatography subunits.
  • the present invention also provides a purification system for continuous purification as described above, which in turn comprises:
  • each affinity chromatography main unit independently contains one or more affinity chromatography subunits
  • At least one virus inactivation main unit wherein the virus inactivation main unit each independently includes one or more virus inactivation subunits;
  • At least one cation exchange chromatography main unit each of which independently includes one or more cation exchange chromatography subunits;
  • At least one anion exchange chromatography main unit each independently comprising one or more anion exchange chromatography subunits;
  • At least one concentration and/or liquid exchange main unit wherein each concentration and/or liquid exchange main unit independently includes one or more concentration and/or liquid exchange sub-units.
  • the present invention also provides a purification system for continuous purification as described above.
  • the management system After at least one sub-unit target liquid of at least one upstream main unit flows out of the main unit, the management system allows At least one subunit of the upstream main unit immediately adjacent to the downstream main unit starts operation; and the management system allows the first subunit target liquid of any one of the upstream main units to flow out of the upstream main unit, so The running time of the upstream main unit and the immediately downstream main unit at least partially overlap.
  • the present invention also provides a purification system for continuous purification as described above, wherein the continuous purification is continuous, closed, and automated separation and purification of target molecules.
  • the present invention also provides a continuous purification method for target molecules, which is completed by the system of the present invention.
  • the method further includes flowing the clarified cell harvest fluid (CCCF) containing the target molecule into the buffer volume immediately upstream of the at least one affinity chromatography main unit as described above, and making the CCCF After the steps of separation and purification by the purification system as described above.
  • CCCF clarified cell harvest fluid
  • the purification system includes an online target protein quantitative detection system.
  • the online target protein quantitative detection system includes an on-site UV absorbance detector, protein concentration calculation equipment and protein load control equipment.
  • the on-site UV absorbance detector detects the UV absorption value of the material and transmits the data to the protein concentration calculation device.
  • the protein concentration calculation device calculates the protein concentration and the total amount of target protein flowing through unit time in real time; and sends the total amount of target protein to the protein load control device (DCS).
  • the online target protein quantitative detection system can judge the loading volume of the chromatography main unit in real time, and control the loading end point of the chromatography main unit, thereby controlling the loading volume.
  • the in-situ UV absorbance detector is a part of the online target protein quantitative detection system.
  • one or more in-situ UV absorbance detectors are configured to communicate with the management system;
  • the management system controls the end point of the sample loading (sample loading process) of the chromatography unit according to the signal detected by the in-situ UV absorbance detector.
  • the on-site UV absorbance detector detects the feed signal of the chromatography unit on-site, which represents the composition of the feed material provided to the inlet of the chromatography unit, and/or
  • the in-situ UV absorbance detector detects the effluent signal of the chromatography unit on-site, which represents the components of the effluent of the chromatography unit.
  • one or more in-situ UV absorbance detectors are arranged upstream of the circulation pipeline of the purification system relative to the at least one chromatography main unit.
  • the in-situ UV absorbance detector includes: a flow cell, an ultraviolet irradiation device, and a spectrometer.
  • the flow cell includes a solution flow channel interface and an optical channel interface.
  • the solution flow channel interface is connected with the chromatography device.
  • the optical channel interface includes a first optical channel interface and a second optical channel interface that are arranged at both ends of the flow cell in a direction perpendicular to the material flow direction, which are respectively connected with the ultraviolet irradiation device and the spectrometer.
  • the spectrometer detects the ultraviolet intensity of the solution containing the target protein irradiated by the ultraviolet irradiation device flowing through the flow cell, and transmits the ultraviolet absorption value data to the protein concentration calculation device.
  • At least one chromatography main unit in the purification system includes a plurality of subunits.
  • a multi-column chromatography system is used to realize multiple subunits; each chromatography column in the multi-column chromatography system is independently located below One of the states: a. loading step, b. non-loading step and c. resting state.
  • one or more in-situ UV absorbance detectors are respectively configured at the upstream and downstream positions of the at least one chromatography unit, which are communicatively connected with the management system.
  • the management system controls the tomographic unit to switch between the states a, b, and c according to the signal detected by the on-site UV absorbance detector.
  • the loading step refers to the contact between the chromatography column packing and the sample for which affinity binding is expected
  • the non-loading step includes: eluting the sample bound to the chromatography column packing, and optionally includes one or more of the following steps: washing impurities of non-target molecules after loading the sample, washing the chromatography after eluting the sample Re-equilibrate the column and clean the chromatographic column;
  • the resting state means that there is no sample in contact with the chromatography column packing and/or there is basically no fluid communication between the chromatography column and other parts of the circulation pipeline of the purification system.
  • the liquid or solute that is not desired to remain in the concentration and/or liquid exchange step is removed in the direction of filtration.
  • the liquid or solute desired to be replaced is added to the stream containing the sample in the direction of filtration.
  • the stream containing the sample flows to the downstream direction of the circulation pipe of the purification system relative to the concentration and/or liquid exchange unit.
  • the concentration and/or liquid exchange step is tangential flow filtration, especially unidirectional tangential flow filtration.
  • the concentration and/or fluid exchange step is unidirectional tangential flow filtration and/or in-situ dialysis.
  • the management system includes elements for data processing.
  • the upstream and downstream of the flow pipeline of the purification system relative to the specific chromatography unit are equipped with one or more UV absorbance detectors in place, and the management system controls the loading of the chromatography unit by the following method End point: the management system uses the feed signal and the effluent signal to determine the binding state of the chromatography medium in the chromatography unit, which includes using the feed signal and the effluent signal to determine the delta signal And the maximum delta signal, the delta signal is the feed signal minus the effluent signal, the maximum delta signal is when the effluent signal has passed through the chromatography medium in the chromatography unit due to substantially all unbound components This fact shows that the feed signal minus the effluent signal during the plateau period;
  • the ⁇ signal is used to determine the penetration point and/or the saturation point of the tomographic unit, and the penetration point and the saturation point are calculated as a corresponding predetermined percentage of the maximum ⁇ signal;
  • the management system issues an instruction to end the sample loading operation.
  • the management system issues instructions based on the binding state of the chromatography media in the chromatography unit (for example, whether the column reaches the penetration point and/or saturation point) and a preset algorithm to control the chromatography unit in the Switch between states a, b, and c.
  • the purification system further comprises one or more virus removal units.
  • the virus removal unit is virus removal filtration, virus inactivation, or a combination thereof.
  • the method of virus inactivation is low pH inactivation.
  • the virus removal unit is configured with a sensor, and the sensor is in communication connection with the management system.
  • the sensor is selected from a pH sensor, a conductivity sensor, and a level sensor.
  • the buffer volume is a buffer tank.
  • the buffer volume and/or virus removal unit is further configured with other sensors, preferably, the other sensors are selected from pressure sensors, optical sensors, and combinations thereof.
  • the management system also issues instructions based on the signals from the sensors and a preset algorithm to control the flow rate of the material between unit operations and/or within the unit operations.
  • the material flows into the specific main unit from the immediately upstream buffer volume or the immediately upstream main unit, and then the main unit performs unit operations and generates Output flow:
  • the output flow flows into the buffer volume immediately downstream or the immediately downstream main unit according to the instruction of the management system; when the unit operation of the specific main unit ends, the generation of the output flow stops.
  • the affinity chromatography main unit, virus removal main unit, concentration and/or liquid exchange main unit in the purification system are distributed from upstream to downstream in the circulation pipeline of the purification system.
  • the feed material in the purification system is the use of host cells to express recombinant protein to obtain CCCF.
  • the CCCF is obtained by at least steps including centrifugation and deep filtration after cell culture.
  • the purification system further comprises one or more additional chromatographic main units, and the chromatography is selected from mixed mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography, hydroxyl group Apatite chromatography, and any combination of the above-mentioned various chromatography.
  • the in-situ UV absorbance detection device of the present invention is configured in the purification system, and its purpose is to provide an online target protein quantitative detection system to realize the online quantitative detection of the target protein during the purification process of the target protein and the control of the target protein according to the detection result.
  • the loading capacity of the target protein is configured in the purification system, and its purpose is to provide an online target protein quantitative detection system to realize the online quantitative detection of the target protein during the purification process of the target protein and the control of the target protein according to the detection result.
  • the online target protein quantitative detection system of the present invention includes:
  • the flow cell includes a solution flow channel interface and an optical channel interface, the solution flow channel interface is connected to the chromatography device, and the optical channel interface is connected to the protein concentration detection and calculation device;
  • a chromatographic device connecting the solution containing the target protein to the online target protein quantitative detection system, and performing protein separation on the solution containing the target protein flowing through to obtain the target protein solution;
  • a protein concentration detection and calculation device which detects the concentration of the target protein-containing solution, and calculates the total amount of protein in the target protein-containing solution in real time;
  • the protein load control device is in communication connection with the protein concentration detection and calculation device and the chromatography device, and controls the sample load of the chromatography device according to the total amount of protein.
  • the chromatography device includes:
  • Chromatography equipment connected to the solution flow channel interface of the flow cell, and connecting the solution containing the target protein to the online target protein quantitative detection system;
  • At least one chromatographic column connected to the solution flow channel interface of the flow cell, performs protein separation on the solution containing the target protein flowing through, and removes other components in the solution containing the target protein to obtain the target protein solution .
  • the protein concentration detection and calculation device includes: an ultraviolet irradiation device connected to the optical channel interface of the flow cell to irradiate the solution containing the target protein flowing through the flow cell;
  • a spectrometer connected to the optical channel interface of the flow cell, detects the ultraviolet intensity of the solution containing the target protein that is irradiated by the ultraviolet irradiation device flowing through the flow cell, and transmits the ultraviolet absorption value data to the protein concentration calculation device ;
  • the protein concentration calculation device receives the ultraviolet absorption value data of the spectrometer, and calculates the protein concentration and the total amount of target protein flowing through the flow cell per unit time in real time according to a preset calibrated standard curve; and the target protein The total amount is sent to the protein load control device.
  • the protein concentration calculation device and the protein load control device in the protein concentration detection calculation device are included in the management system in the purification system for continuous purification of the present invention.
  • the flow cell and the ultraviolet irradiation device and the spectrometer in the protein concentration detection and calculation device and the connection between them are the in-situ UV absorbance detector in the purification system of the present invention.
  • the protein load control device includes:
  • the sample loading control module compares the total amount of the target protein received as the cumulative sample load with the preset sample load of the chromatography column in real time, and compares the cumulative sample load with the preset total sample load. When the loading capacity of the chromatography column is equal, control the chromatography equipment to stop loading; and
  • the selection and calling module executes the control operations of selecting and calling the preset calibrated standard curve, UV zero signal, target protein accumulation command, and target protein quantitative accumulation upper limit setting.
  • the protein concentration calculation device is an industrial computer
  • the protein load control device is a DCS system.
  • the solution flow channel interface of the flow cell includes a solution inflow interface and a solution outflow interface that are arranged at both lateral ends of the flow cell;
  • the optical channel interface includes an upper interface that is arranged at both longitudinal ends of the flow cell And the lower end interface, and in the middle of the flow cell there is an optical aperture for the solution containing the target protein to pass through;
  • the solution inflow port of the flow cell is connected to the chromatography device, the solution outflow port of the flow cell is connected to the chromatography column; the upper port is connected to the ultraviolet irradiation device, and the lower port is connected to the chromatographic column.
  • the spectrometer connection is connected.
  • the flow cell is a variable optical path flow cell; the optical path of the variable optical path flow cell is adjusted by adjusting the size of the optical path opening;
  • the optical aperture is formed by the distance between two optical fibers arranged in the middle of the flow cell, and the distance between the two optical fibers is 0.1 mm to 10 mm.
  • the upper end interface and the lower end interface are optical fiber interfaces
  • the two optical fibers are connected to the flow cell through the upper end interface and the lower end interface
  • the optical fiber connected to the upper end interface is connected to the The light-emitting light source of the ultraviolet irradiation device is connected
  • the optical fiber connected to the lower end interface is connected to the spectrometer.
  • the optical fiber is an anti-ultraviolet quartz optical fiber, and the core diameter of the optical fiber is 450 microns; the two optical fibers are connected to the flow cell through an SMA905 joint.
  • the light source of the ultraviolet irradiation device includes a xenon lamp, a deuterium lamp, an LED lamp or a mercury lamp; the emission wavelength of the light source of the ultraviolet irradiation device is 200nm-400nm;
  • the spectrometer includes: an ultraviolet spectrometer and an ultraviolet-visible spectrometer; the absorbance range of the spectrometer is 0-2 AU.
  • the wavelength range of the emitted light of the ultraviolet irradiation device is 270nm-320nm;
  • the absorbance range of the spectrometer is 0.05AU-1.6AU;
  • the target protein is a monoclonal antibody.
  • the online target protein quantitative detection system is configured to include: a flow cell, a chromatography device, a protein concentration detection calculation device and a protein load control device.
  • the flow cell includes a solution flow channel interface and an optical channel interface, the solution flow channel interface is connected to the chromatography device, the optical channel interface is connected to the protein concentration detection and calculation device; the chromatography device connects the solution containing the target protein to the
  • the online target protein quantitative detection system is used for protein separation of the solution containing the target protein flowing through, and the protein concentration detection calculation device detects the concentration of the target protein-containing solution, and calculates the concentration of the target protein-containing solution in real time
  • the protein load control device is communicatively connected with the protein concentration detection and calculation device, and the sample load of the chromatography device is controlled according to the total amount of protein, thereby realizing online quantitative detection of the target protein And control the loading capacity of the target protein according to the detection result.
  • the performance of the spectrometer used in the present invention is better, the shortest integration time is 10 microseconds, the response is faster, it is more suitable for the needs of online detection, and the deuterium lamp emits more stable.
  • the invention adopts a flow cell to realize the optical path change of 0.1-10mm, the structure is simpler, and the measurable concentration range is wider.
  • the SMA-905 interface is adopted as the standard interface, which has strong adaptability; when the material liquid passes through the flow cell, since the material liquid flowing through the optical fiber slit is very small, most of it passes through the external space without causing resistance.
  • the use of industrial communication protocols between multiple devices is more stable and reliable; and the present invention provides more online control functions.
  • the online HPLC device includes a sampling line, a sample loop, a six-way valve, an HPLC equipped with a pump and an analytical column, and a detector, and the pump drives the flow/stop of the sample in the online HPLC device.
  • the sampling pipeline is a branch on the eluent outlet pipeline of the chromatography equipment. The sample enters the sample loop in the six-way valve through the sampling pipeline, and then returns to the eluent outlet pipeline.
  • the online HPLC device sends a signal to the DCS and is controlled by the DCS.
  • the six-way valve switches the loop, and the HPLC pump pushes the sample in the sample loop onto the HPLC column.
  • the results are transmitted to the DCS system.
  • the DCS system switches the corresponding valve according to the result, and guides the sample combination according to the sample standard.
  • the detector of the online HPLC device is an ultraviolet-visible absorption detector, such as an ultraviolet absorption detector or a DAD detector.
  • the purification system of the present invention includes an operation layer and an automation layer.
  • the operating layer includes all process technology elements (PTU) and service technology elements (STU), such as the plurality of chromatography main units, optionally one or more concentration and/or liquid exchange main units, and optionally one Or multiple virus removal main units, optionally one or more buffer volumes, and hoses, filters, chromatography columns, containers, sensors, pumps, valves, etc. present in the units.
  • PTU process technology elements
  • STU service technology elements
  • the operation layer consists of three layers connected in the following order: (1) Material input layer; (2) Convergence layer; (3) Product output layer.
  • the raw material input layer inputs the raw materials to be chromatographed into the system according to the supply demand. It should be understood that since the common production processes of biomolecules are based on processes involving the use of living organisms or cultured cells, these processes are more difficult to control than chemical synthesis, and the expected homogeneity of the materials produced is relatively low, so different time points The difference between the raw materials input to the purification system is relatively high. In the space composed of parameters used to characterize the properties of materials, the properties of the raw materials input to the purification system at different time points are distributed at multiple points.
  • the material input to the convergence layer has undergone at least one purification step, such as a chromatographic step. Therefore, in the space composed of parameters used to characterize the properties of the material, the degree of dispersion of the attributes of the material input to the convergence layer at different time points is relatively In terms of raw materials, it has been reduced and has been converged in one or more dimensions (ie, one or more parameters); and (2) According to the pre-set parameter requirements, the convergent layer puts the materials on the basis of (1) Further convergence is performed so that the materials output by the convergence layer have higher convergence properties in one or more preset dimensions (ie, one or more parameters) than the materials input to the convergence layer.
  • the output material of the convergence layer contains substantially all or a part of the target molecules contained in the raw material.
  • the number of chromatographic subunits that need to be configured shows a tendency to decrease compared to the upstream of the convergence layer.
  • N represents the number of chromatography subunits included in a certain chromatography main unit.
  • the trend of reducing the number of chromatography subunits can be expressed by the following formulas (1) and/or (2) :
  • N convergence layer
  • ⁇ N adjacent chromatographic main unit upstream of the convergence layer
  • the purification main unit adjacent to the downstream of the convergence layer is a chromatography main unit, a virus removal main unit or a concentration and/or liquid exchange main unit.
  • At least one convergence layer is feedback controlled by PAT (see “PAT for Feedback Control” below for details).
  • the product output layer outputs products from the purification system according to supply requirements.
  • the product output by the purification system of the present invention can meet the product quality requirements in some cases, preferably in most cases, and more preferably basically every time it is output.
  • the product quality requirements refer to the requirements specified by technical requirements and regulatory specifications for products at this stage (for example, raw liquid, semi-finished products, or finished products).
  • the operation layer includes a raw material input layer, at least one convergence layer, and a product output layer. In one embodiment, the operation layer includes a raw material input layer, one or two convergence layers, and a product output layer.
  • the materials Before the materials enter the convergence layer, they can be combined by fractions, and after the materials flow out of the chromatographic unit in the convergence layer, the materials can optionally be further combined by fractions.
  • the merging of fractions refers to merging samples with specific attributes that meet certain requirements.
  • the accumulation of materials by the accumulation layer is achieved by chromatographic purification and subsequent fraction merging.
  • the convergence layer also plays a role in reconciling the difference in process cycle time between steps.
  • the management system can know the distribution of materials, and thereby determine the start or stop of one or more steps in the entire process, and ultimately minimize the production time. Therefore, the convergence layer also realizes the convergence of operating time, namely the following point III:
  • the chromatographic main unit in the convergence layer contains a chromatographic subunit
  • the chromatographic main unit in the convergence layer contains multiple chromatographic subunits; the operating time of multiple chromatographic subunits is mutually exclusive
  • the purification system includes a first chromatography main unit, a virus removal main unit, a second chromatography main unit, a third chromatography main unit, and a concentration and/or liquid exchange main unit connected in sequence; the first layer
  • the output stream of the analysis main unit is used as the input stream of the virus removal main unit
  • the output stream of the virus removal main unit is used as the input stream of the second chromatography main unit
  • the output stream of the second chromatography main unit is used as the third chromatography main unit.
  • Input stream; the output stream of the third chromatography main unit is used as the input stream of the concentration and/or liquid exchange main unit.
  • the first chromatographic main unit includes 7 subunits, which are realized by three chromatography columns, and the virus removal main unit includes 3 subunits, which are realized by running a low-pH virus inactivation tank for three cycles.
  • the second chromatographic main unit contains 3 sub-units, which are realized by running a chromatography column for three cycles, and the third chromatographic main unit contains 1 sub-unit.
  • the virus removal main unit starts the first operation cycle, and the operations of the third and fourth subunits of the first chromatography main unit are completed.
  • the virus removal main unit starts the second operation cycle, and after the operations of the fifth, sixth, and seventh subunits of the first chromatography main unit are completed, the virus removal main unit starts the third operation cycle.
  • the operation time of one operation cycle of the virus removal main unit is shorter than the operation time of the two subunits of the first chromatography main unit.
  • the first chromatography main unit performs chromatography in a bind-elution mode.
  • the second chromatography main unit starts the first operation cycle.
  • the waiting time t1 passes, and the second layer
  • the analysis main unit starts the second operation period
  • the second chromatography main unit starts the third operation period after the waiting time t2 has elapsed.
  • the first, second, and third operation periods of the second chromatography main unit are completely continuous in time, and t1 and t2 are respectively equal to the time when the second or third operation period of the second chromatography main unit starts. Reduce the time for the end of the second or third operation cycle of the virus removal main unit.
  • the first chromatography main unit is an affinity chromatography main unit
  • the second and third chromatography main units are respectively ion exchange chromatography main units.
  • the second chromatography main unit performs chromatography in a bind-elution mode.
  • the third chromatography main unit performs chromatography in a flow-through mode.
  • the main unit of affinity chromatography is located upstream of the pooling layer.
  • the convergence layer comprises a cation exchange chromatography main unit, an anion exchange chromatography main unit, or a combination thereof.
  • the main unit for concentration and/or liquid exchange is located downstream of the pooling layer.
  • the purification system sequentially includes: an affinity chromatography main unit, a cation exchange chromatography main unit, and an anion exchange chromatography main unit, wherein the cation exchange chromatography main unit is a convergent layer, and the number of convergent layers is one.
  • N denote the number of chromatographic subunits included in a certain chromatographic main unit, and the convergence effect of the convergent layer reduces the trend of the number of chromatographic subunits can be expressed by the following formulas (1) and (2):
  • the purification system sequentially includes: an affinity chromatography main unit, a cation exchange chromatography main unit, and an anion exchange chromatography main unit, wherein the cation exchange chromatography main unit is the aggregation layer 1, and the anion exchange chromatography main unit It is aggregation layer 2, and the number of aggregation layers is 2.
  • N represent the number of chromatographic subunits included in a certain chromatographic main unit, and the convergence effect of the convergent layer reduces the trend of the number of chromatographic subunits can be expressed by the following formula (3):
  • the automation layer includes components for data collection and processing.
  • the automation layer refers to RIO (ie, an industrial computer or a PC-based system with an I/O layer, or a combination thereof).
  • the industrial computer is a programmable logic control (PLC).
  • PLC programmable logic control
  • the automation layer refers to a DCS system with an I/O layer.
  • the automation layer is a combination of an industrial computer and a DCS system with an I/O layer.
  • the management system of the purification system of the present application refers to an industrial computer and a DCS system.
  • the DCS system uses multiple communication protocols to communicate with devices, examples of which include, but are not limited to, Profibus-DP, OPC, Modbus-RTU, 4-20 mA signals, and the like.
  • the control program runs in the main controller or the sub-controller of the DCS system. The data generated by the actuator or sensor is collected by the DCS system and stored.
  • PAT is used for feedback control
  • the device of the present invention for realizing PAT is the in-situ UV absorbance detector of the present invention.
  • the device of the present invention for realizing PAT is a combination of the in-situ UV absorbance detector of the present invention and other in-situ or online detection technologies.
  • the other in-situ or online detection technology is selected from: online or in-situ spectroscopy methods (such as near infrared (NIR), mid-infrared (MIR) or Raman spectroscopy, etc.), online or in-situ chromatography Methods (e.g. online chromatography, such as online HPLC) and combinations thereof.
  • the other in-situ or online detection technology is online HPLC.
  • At least one of the devices for implementing PAT of the present invention is configured at the convergence layer.
  • the management system controls one or more of the following operations based on the signal obtained by the device for implementing PAT:
  • the output stream from the upstream unit or the material in the buffer volume flows out as the input stream into the convergence layer;
  • the output stream from the upstream unit included in the convergence layer flows into the downstream unit as the input stream of the downstream unit included in the convergence layer;
  • the output stream from the upstream unit included in the convergence layer flows into the buffer volume between the upstream unit and the downstream unit as the downstream unit input stream included in the convergence layer;
  • a schematic diagram of the operation layer including a convergence layer is shown in Fig. 1, in which one or more of operations D, E, F, G, and H are controlled by the management system according to the signal obtained by the device for implementing PAT .
  • the convergence layer is configured with at least one device for implementing PAT.
  • the convergence layer is configured with at least one in-situ UV absorbance detector of the present invention.
  • the convergence layer is also equipped with an online HPLC device.
  • the convergence layer includes at least one chromatographic unit, at least one on-site UV absorbance detector is arranged upstream of the at least one chromatographic unit, and the management system determines the chromatographic unit in accordance with the method described above. The loading capacity of the chromatography medium.
  • the sample loading condition of the chromatography medium in the chromatography subunit it is determined whether it is necessary to add a new chromatography subunit to the chromatography main unit to which the chromatography subunit belongs to load samples and perform purification. , And make the total running time span of the chromatography main unit fall within a preset range.
  • micro equipment and integrated devices can be used.
  • large-scale implementation such as industrial production and preparation, large-scale chromatography towers, chromatography beds and other equipment can be used.
  • the purification system of the present invention includes a buffer volume 1, an affinity chromatography main unit, a buffer volume 2, a virus removal main unit 1 (virus removal tank), a buffer volume 3, and a cation exchange chromatography.
  • FIG. 2 the purification system of the present invention is shown in FIG. 2.
  • the actuators for operating the material to flow into/out of the buffer volume are the transfer pump, the bottom valve of each buffer tank and the virus removal tank.
  • Each buffer volume is equipped with a weight sensor and a liquid level sensor.
  • the virus removal tank is equipped with a pH sensor, a conductivity sensor, a weight sensor and a liquid level sensor.
  • Each buffer volume and virus removal tank are independently set with a lower limit of material liquid and an upper limit of material liquid (measured by the detection value of the weight sensor or the detection value of the weight sensor and the liquid level sensor).
  • the buffer volume is considered to be unsuitable for supplying the material to the downstream main unit, thereby stopping the transfer of the material to the main unit and When appropriate, stop the operation of the downstream main unit; when the value of the weight sensor reaches or exceeds the upper limit of weight, it is considered that the buffer volume is no longer suitable to accept the feed liquid input by the upstream main unit, thereby stopping the operation of the upstream main unit.
  • a material liquid detection interval is independently set to determine whether the immediately downstream main unit can be activated.
  • This weight interval is called the interval of the immediately downstream main unit that activates the buffer volume, and is referred to as the buffer for short.
  • the starting interval of the volume The lower limit of the starting interval is the lower limit of the buffer volume, and the upper limit of the starting interval is less than or equal to the upper limit of the buffer volume.
  • the purification process can be described as the following steps 1-8:
  • the clarified cell harvest liquid used as raw material is temporarily stored in buffer volume 1;
  • Buffer volume 1 and buffer volume 2 weight and liquid level control affinity chromatography main unit:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 1 into the affinity chromatography main unit;
  • the material flows into the buffer volume 2 from the main unit of affinity chromatography;
  • the DCS system sends a Digital I/O signal to the PCC. After receiving the signal, the PCC ends the sample loading process and enters the end step. After the PCC program ends, the operation of the affinity chromatography unit is completed;
  • the DCS controls the PCC to suspend operation. After the suspension is suspended until the weight of the buffer volume 2 is within the starting range again, the PCC continues to run;
  • Buffer volume 2 virus removal main unit 1 and buffer volume 3
  • Weight and liquid level control virus removal main unit 1 Weight and liquid level control virus removal main unit 1:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 2 into the virus removal main unit 1;
  • the DCS system judges whether the pH value of the virus removal main unit 1 is within the required range, and then automation is required as needed Controlled pump operation adds acid or alkali to inactivate low pH virus;
  • the material flows from the virus removal main unit 1 (virus removal main unit 1) into the buffer volume 3 through deep filtration;
  • the transfer of materials to the buffer volume 3 is ended; the virus removal main unit 1 is cleaned;
  • the DCS controls the suspension of the transfer of materials to the virus removal main unit 1, and the material continues to be transferred to the virus removal main unit 1 after the suspension is suspended until the weight of the buffer volume 3 is within the starting interval again.
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 3 into the main unit of cation exchange chromatography.
  • the DCS system sends instructions to start the main unit of cation exchange chromatography.
  • the DCS system judges whether the conductivity value of the buffer volume 3 is within the set target range according to the conductivity sensor data, and conducts conductivity adjustment operations as needed , Make the conductivity value reach ⁇ 0.1-0.5mS/cm of the set range, stable for 1-5min.
  • the DCS system sends a start protein accumulation instruction to the online target protein quantitative detection system, starts real-time calculation of the loaded protein amount, and sends the calculation result to the DCS system.
  • the DCS system gives Process sends instructions to stop sample loading and enter the subsequent chromatography steps.
  • the online target protein quantitative detection system still does not reach the protein cumulative set value, then the buffer volume 3 is judged to be empty, and the DCS system will give you Process sends instructions to stop sample loading and enter the subsequent chromatography steps.
  • the material flows into the buffer volume 4 from the main unit of cation exchange chromatography.
  • Buffer volume 4 buffer volume 5 weight and liquid level and Process control anion exchange chromatography main unit:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 4 into the anion exchange chromatography main unit.
  • the DCS program makes a judgment based on the online pH and conductivity sensor data, and adjusts the pH and conductivity as needed.
  • the DCS system sends an instruction to start the protein amount accumulation instruction to the online target protein quantitative detection system, starts to calculate the sample protein amount in real time, and sends the calculation result to the DCS system.
  • the DCS system gives Process sends instructions to stop sample loading and continue the chromatography step. If the weight of the buffer volume 4 is lower than the lower limit, the online target protein quantitative detection system still does not reach the protein cumulative set value, then the buffer volume 4 is judged to be empty, and the DCS system will give you Process sends instructions to stop sample loading and continue the chromatography step.
  • the material flows into the buffer volume 5 from the main unit of anion exchange chromatography.
  • Buffer volume 5 buffer volume 6 weight and liquid level, and constant pressure control of virus removal main unit 2:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 5 into the virus removal main unit 2 for virus removal filtering.
  • the virus-removing filtration pressure is controlled to a constant pressure.
  • the material flows from the virus-removing main unit 2 into the buffer volume 6.
  • the DCS controls the suspension of the transfer of materials to the virus removal main unit 2. After the suspension is suspended until the weight of the buffer volume 6 is within the starting interval again, the material transfer to the virus removal main unit 2 is continued.
  • Buffer volume 6 buffer volume 7
  • weight and liquid level weight and liquid level, and concentration and/or liquid exchange main unit 1 constant pressure control:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 6 into the main unit 1 for concentration and/or liquid exchange.
  • the pump flow rate is adjusted to control the pressure to be constant.
  • the DCS controls the pump to suspend operation. After the suspension is suspended until the weight of the buffer volume 7 is within the starting interval again, the pump continues to run.
  • the material flows into the buffer volume 7 from the concentration and/or liquid exchange main unit 1.
  • Buffer volume 7, buffer volume 8, weight and liquid level, and concentration and/or liquid exchange main unit 2 constant pressure control:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 7 into the concentration and/or liquid exchange main unit 2.
  • the pressure at the inlet of the tangential flow exchange unit is controlled to be a constant pressure.
  • the material flows into the buffer volume 8 from the concentration and/or liquid exchange main unit 2, and is output as a product.
  • the stream flowing from the buffer volume 1 into the affinity chromatography main unit contains one or more sub-streams
  • the stream flowing into the buffer volume 2 from the affinity chromatography main unit contains multiple sub-streams
  • the material flow from the buffer volume 2 into the virus removal main unit 1 contains a sub-flow
  • the stream flowing into the buffer volume 3 from the virus removal main unit 1 contains multiple sub-streams
  • the stream flowing from the buffer volume 3 into the main unit of cation exchange chromatography includes a sub-stream
  • the stream flowing into the buffer volume 4 from the cation exchange chromatography main unit contains one or more sub-streams
  • the stream flowing from the buffer volume 4 into the anion exchange chromatography main unit contains a sub-stream
  • the stream flowing into the buffer volume 5 from the anion exchange chromatography main unit contains a sub-stream
  • the material flow from the buffer volume 5 into the virus removal main unit 2 includes a sub-flow
  • the flow from the virus removal main unit 2 into the buffer volume 6 contains a sub-flow
  • the stream flowing from the buffer volume 6 into the concentration and/or liquid exchange main unit 1 contains a sub-stream
  • the stream flowing into the buffer volume 7 from the concentration and/or liquid exchange main unit 1 contains a sub-stream
  • the stream flowing from the buffer volume 7 into the concentration and/or liquid exchange main unit contains a substream 2;
  • the stream flowing into the buffer volume 8 from the concentration and/or liquid exchange main unit 2 contains a sub-stream.
  • the present invention provides an integrated, automated, and fully continuous monoclonal antibody purification system, which is characterized in that (1) an automatic control system is used to communicate with all the equipment to realize the adjustment and control of the process. (2) Realize the full continuous production of the entire purification process of affinity chromatography, low pH virus inactivation, cation exchange chromatography, anion exchange chromatography, virus removal filtration, ultrafiltration concentration, and ultrafiltration liquid exchange. (3) PAT is used to detect the key quality attributes in the production process on-site or online, and realize feedback control through the automatic control system.
  • the present invention realizes PAT with a simple, low-cost, easy-to-implement and maintain online target protein quantitative detection system.
  • the in-place UV absorbance detection equipment uses a flow cell to achieve a 0.1-10mm optical path change, with a simpler structure, a relatively large variable optical path range, and a wider range of measurable concentrations.
  • the online target protein quantitative detection system of the present invention calculates the total amount of target protein flowing through the unit time in real time. It is optionally configured in the same chromatographic main unit with the online HPLC device, and can provide information related to the sample amount and product purity in time.
  • online quantitative detection of the target protein is realized, and the loading capacity of the target protein is controlled according to the detection result, and the purification parameters are adjusted in time through feedback control to ensure product quality when needed.
  • the present invention controls the start and stop of the main unit by specifying the weight of the buffer tank upstream of the main unit, and realizes the automatic unit operation without the need to know the total amount of material liquid in the upstream and downstream tanks.
  • a buffer tank is installed upstream of the affinity chromatography main unit, which can achieve weight control while setting weight protection to avoid damage to the chromatography column caused by the pump inhaling air when the material liquid is exhausted.
  • the weight control method is more stable and reliable than the conventional method of using bubble induction to control the end point of the sample.
  • the method of using bubble induction to control the end point of the sample is often caused by the small bubbles in the clarified cell harvest solution (due to the fact that the fluid is in the Collision in the pipeline, the presence of air in the pipeline, or the existence of dead angles in the pipeline, etc.), leading to the premature end of the sample loading, there is a certain risk.
  • the complex raw materials can be quickly judged and processed to ensure that the CM unit operation can continuously output products with reliable quality attributes, and the subsequent chromatography steps
  • a relatively simple process is achieved.
  • the optional use of online HPLC to detect the content of charge isomers achieves a more complete control of product quality.
  • the entire process control process is relatively simple, has high stability, and is suitable for a wide range of process conditions.
  • the purification time of the target molecule can be greatly shortened, and the purification efficiency can be greatly improved.
  • the risk of product contamination is greatly reduced, and the space occupied by the biopharmaceutical purification equipment is increased. Utilization rate reduces the space required for equipment, greatly reduces production costs and increases productivity.
  • the PCC system comes from GE Healthcare.
  • Affinity chromatography packing (MabSelect Sure LX) is from GE.
  • the cation exchange chromatography packing (model: Capto S) comes from GE.
  • Anion exchange chromatography packing (model: Capto Q) comes from GE.
  • the SPTFF membrane package is from Pall.
  • the ILDF membrane package is from Pall.
  • the online HPLC equipment is from Agilent, model 1260, equipped with a UV detector.
  • the other reagents of the present invention are all commercially available, for example, they can be purchased from Merck Chemicals.
  • the purification system of the present invention can be implemented in different ways.
  • An exemplary method is as follows: a purification system for continuous purification of monoclonal antibodies, which can be implemented as shown in Figure 2:
  • the purification system of this embodiment includes the buffer volume 1 to the concentration and/or liquid exchange main unit 2 shown in FIG. 2, and includes the material flow processes shown in ii to xv in FIG. 2.
  • the process in Figure 2 can be described as the following steps 1-8, in which the actuators for operating the material to flow into/out of the buffer volume are the transfer pump and the bottom valve of each tank.
  • Each buffer volume is equipped with a weight sensor and a liquid level sensor.
  • the virus removal tank (the main virus removal unit 1) is equipped with a pH sensor, a conductivity sensor, a weight sensor and a liquid level sensor.
  • Each buffer volume and virus removal tank are independently set with a lower limit of material liquid and an upper limit of material liquid (measured by the detection value of the weight sensor or the detection value of the weight sensor and the liquid level sensor).
  • the buffer volume is considered to be unsuitable for supplying the material to the downstream main unit, thereby stopping the transfer of the material to the main unit and When appropriate, stop the operation of the downstream main unit; when the value of the weight sensor reaches or exceeds the upper limit of weight, it is considered that the buffer volume is no longer suitable to accept the feed liquid input by the upstream main unit, thereby stopping the operation of the upstream main unit.
  • a material liquid detection interval is independently set to determine whether the immediately downstream main unit can be activated.
  • This weight interval is called the interval of the immediately downstream main unit that activates the buffer volume, and is referred to as the buffer for short.
  • the starting interval of the volume The lower limit of the starting interval is the lower limit of the buffer volume, and the upper limit of the starting interval is less than or equal to the upper limit of the buffer volume.
  • the raw material CCCF is temporarily stored in buffer volume 1 (Tank0), i.e. i in the figure.
  • Tank0 and buffer volume 2 (Tank1) weight and level control affinity chromatography main unit:
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from the buffer volume 1 to the affinity chromatography main unit through one or more substreams, which is ii in the figure.
  • the PCC program is started by the DCS system to run the affinity chromatography method.
  • the material flows into the buffer volume 2 (Tank1) from the affinity chromatography main unit through multiple substreams, which is iii in the figure.
  • the DCS system sends a Digital I/O signal to the PCC. After receiving the signal, the PCC ends sample loading and enters the end step. After the PCC program ends, the operation of the affinity chromatography unit is completed.
  • the DCS controls the PCC to suspend operation. After the suspension is suspended until the weight of Tank1 is within the starting range again, the PCC continues to operate.
  • Tank2 the management system issues an instruction to instruct the actuator to operate so that the material flows from the buffer volume 2 through a subflow into the virus removal main unit 1 (Tank2), which is iv in the figure.
  • Tank2 When the weight of Tank2 reaches the set lower limit, start mixing. After Tank1 transfers to Tank2, it is stable for 1-2 minutes. According to the pH sensor data, the DCS system judges whether the pH value of Tank2 is within the required range, and then adds acid or alkali according to the needs of the automatic control pump operation. Perform low pH virus inactivation operations.
  • Tank2 After the low pH virus inactivation operation of Tank2 is completed, the stirring is stopped, and the material flows from the virus removal main unit 1 (Tank2) through one or more substreams into the buffer volume 3 (Tank3), which is v in the figure.
  • Tank2 When the weight of Tank2 is lower than the lower limit, the transfer of materials to Tank3 is ended, the cleaning pump is started, and Tank2 is cleaned.
  • DCS controls the suspension of the transfer of materials to the virus removal main unit 1. After the suspension is suspended until the weight of Tank3 is within the starting range again, the material continues to be transferred to the virus removal main unit 1.
  • the DCS system determines whether the tank3 conductivity value is within the set target range, and conducts conductance adjustment operations as needed to make the conductivity value reach ⁇ of the set range 0.1-0.5mS/cm, stable for 1-5min.
  • the stirring is stopped, and the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 3 through a sub-flow into the main unit of cation exchange chromatography, which is vi in the figure.
  • the DCS system sends instructions to start the main unit of cation exchange chromatography.
  • the DCS system sends an instruction to start the protein amount accumulation, and the protein concentration calculation device starts to calculate the sample protein amount in real time, and sends the calculation result to the DCS system.
  • the DCS system gives Process sends instructions to stop sample loading and enter the subsequent chromatography steps (such as elution).
  • Tank3 If after the weight of Tank3 is lower than the lower limit, the online target protein quantitative detection system still does not reach the protein cumulative set value, then it will be judged that Tank3 is empty, and the DCS system will give you Process sends instructions to stop sample loading and enter the subsequent chromatography steps.
  • the material flows into the buffer volume 4 (Tank4) from the cation exchange chromatography main unit through one or more substreams, which is vii in the figure.
  • Tank4 If the weight of Tank4 reaches the upper limit of weight, it will be controlled by DCS Process pauses the cation exchange chromatography operation, and pauses until the Tank4 weight is within the start range again, Process continues to run.
  • Tank4 Tank5 weight and liquid level and Process control anion exchange chromatography main unit:
  • the DCS program judges based on the online pH and conductivity sensor data, and adjusts the pH and conductivity as needed.
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from the buffer volume 4 through a sub-flow into the main unit of anion exchange chromatography, which is viii in the figure.
  • the DCS system sends an instruction to start the accumulation of protein amount, and the protein concentration calculation device starts to calculate the amount of loaded protein in real time, and sends the calculation result to the DCS system.
  • the DCS system gives Process sends instructions to stop sample loading and continue the chromatography step. If after the weight of Tank4 is lower than the lower limit, the online target protein quantitative detection system still does not reach the protein cumulative set value, then it is judged that Tank4 is empty, and the DCS system will give you Process sends instructions to stop sample loading and continue the chromatography step.
  • the material flows into the buffer volume 5 (Tank5) from the main unit of anion exchange chromatography through a substream, which is ix in the figure.
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 5 through a sub-flow into the virus removal main unit 2 for virus removal filtering, which is x in the figure. .
  • the virus-removing filtration pressure is controlled to a constant pressure.
  • the material flows from the virus-removing main unit 2 through a sub-flow into the buffer volume 6 (Tank6), which is xi in the figure.
  • the DCS controls the suspension of the transfer of materials to the virus removal main unit 2. After the suspension is suspended until the Tank6 weight is within the starting interval again, the transfer of materials to the virus removal main unit 2 is continued.
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from the buffer volume 6 through a subflow into the concentration and/or liquid exchange main unit 1, as shown in the figure xii. Start the pump to start the concentration. During the concentration process, the pump flow rate is adjusted to control the pressure to be constant.
  • the DCS controls the pump to suspend operation. After the suspension is suspended until the weight of Tank7 is within the starting range again, the pump continues to operate.
  • the material flows from the concentration and/or liquid exchange main unit 1 into the buffer volume 7 (Tank7) through a substream, which is xiii in the figure.
  • Tank7, Tank8 weight and liquid level, concentration and/or liquid exchange main unit 2 constant pressure control:
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 7 through a subflow into the concentration and/or liquid exchange main unit 2, which is xiv in the figure.
  • the pressure at the inlet of the tangential flow exchange unit is controlled to be a constant pressure.
  • the material flows into the buffer volume 8 (Tank 8) from the main unit 2 for concentration and/or liquid exchange through a sub-flow, and is output as a product, which is xv in the figure.
  • the management system of the purification system includes industrial computer and DCS system.
  • the purification system of the above-mentioned continuous purification of monoclonal antibodies is controlled as a whole by the distributed control system (DCS), which uses a variety of communication protocols to communicate with the equipment, including Profibus-DP, OPC, Modbus-RTU, 4-20mA signal, etc.
  • DCS distributed control system
  • the control program runs in In the main controller or sub-controller of the DCS system.
  • the data generated by the equipment including pumps, agitators, pH sensors, pressure sensors, UV sensors, conductivity sensors, weight sensors, liquid level sensors, etc.
  • the DCS system determines the operation status of the steps, the loading status of the chromatography medium and the preset algorithm, and sends instructions to the actuators (such as pumps or valves) to promote the flow of materials. Flow or stop in the flow path.
  • the actuators such as pumps or valves
  • In-place UV absorbance detectors include: flow cell, ultraviolet irradiation device and spectrometer.
  • the flow cell includes a solution flow channel interface and an optical channel interface. Refer to Figure 3 to Figure 5 for the configuration of the in-situ UV absorbance detector in the purification system.
  • FIGS 3 to 5 show:
  • the flow cell 10 includes a horizontal solution flow channel interface (11, 12) and a longitudinal optical channel interface (13, 14), the solution flow channel interface (11, 12) and the chromatography device 20, the optical channel interface (13,14) Connected to the protein concentration detection and calculation device 30; the flow cell 10 is connected to more than 100 devices of the online target protein quantitative detection system, and the solution containing the target protein passes through the flow cell from the front-end equipment of the system 100 to the system 100 Back-end equipment;
  • the chromatography device 20 connects the solution containing the target protein to the online target protein quantitative detection system 100, and performs protein separation on the solution containing the target protein flowing through to obtain the solution containing the target protein; Other components in the solution containing the target protein are removed, and the other components in the solution containing the target protein include some impurities of other proteins;
  • the protein concentration detection and calculation device 30 detects the concentration of the solution containing the target protein, and calculates the total amount of protein in the solution containing the target protein in real time;
  • the protein load control device 40 is in communication connection with the protein concentration detection and calculation device 30 and the chromatography device 20, and controls the sample load of the chromatography device 20 according to the total amount of protein. After calculating the total amount of protein in the solution containing the target protein, the protein load control device 40 will control the sample load of the chromatography device 20 according to the received information. Specifically, after the total amount of protein in the solution containing the target protein reaches the preset value, the solution loading of the target protein is stopped, thereby realizing the online quantitative detection of the target protein by the online target protein quantitative detection system 100.
  • the target protein is a monoclonal antibody (hereinafter referred to as "monoclonal antibody"). That is, the online target protein quantitative detection system 100 realizes online monoclonal antibody quantitative detection.
  • the chromatography device 20 includes:
  • the chromatography device 21 is connected to the solution flow channel interface (11, 12) of the flow cell 10, and connects the solution containing the target protein to the online target protein quantitative detection system 100;
  • At least one chromatographic column 22 is connected to the solution flow channel interface of the flow cell 19 (11, 12), and performs protein separation on the solution containing the target protein flowing through, and separates the other in the solution containing the target protein The components are removed to obtain the target protein solution.
  • the chromatographic column 22 may include a plurality of chromatographic columns 22, which are arranged at different positions.
  • the flow cell 10 When used to detect the protein concentration before entering the chromatographic column 22 and control the loading amount, the flow cell 10 is arranged at the upstream of the chromatographic column 22 in the flow pipe. When it is needed to detect the protein concentration in the solution flowing out of the chromatography column 22, the flow cell 10 can be placed at a position downstream of the chromatography column 22.
  • the chromatographic device 20 pipeline for example, GE Equipment
  • the protein loading control device 40 will control the loading capacity of the chromatography device 20 according to the received information, specifically controlling the loading capacity of the chromatography column 22 during the monoclonal antibody chromatography.
  • the protein concentration detection and calculation device 30 of the present invention includes: an ultraviolet irradiation device 31, which is connected to the optical channel interface (upper interface) 13 of the flow cell 10, and irradiates the target containing target flowing through the flow cell 10 Protein solution;
  • the light source of the ultraviolet irradiation device 31 includes a xenon lamp, a deuterium lamp, an LED lamp or a mercury lamp;
  • the emission wavelength of the light source of the ultraviolet irradiation device 31 is 200nm-400nm;
  • the spectrometer 32 is connected to the optical channel interface (lower end interface) 14 of the flow cell 10, and absorbs the ultraviolet radiation device 31 to irradiate the ultraviolet rays of the solution containing the target protein flowing through the flow cell 10, and combine the
  • the ultraviolet absorption value data of the spectrometer 32 is transmitted to the protein concentration calculation device 33; the ultraviolet irradiation device 31 irradiates the solution containing the target protein, the solution containing the target protein absorbs ultraviolet rays, and the spectrometer 32 detects that the solution containing the target protein absorbs ultraviolet rays
  • the ultraviolet absorption value data is transmitted to the protein concentration calculation device 33; the spectrometer 32 includes: an ultraviolet spectrometer 32 and an ultraviolet-visible spectrometer 32; the absorbance range of the spectrometer 32 is 0-2 AU.
  • the wavelength range of the emitted light of the ultraviolet irradiation device 31 is 270nm-320nm; the absorbance value of the spectrometer 32 is in the range of
  • the protein concentration calculation device 33 receives the ultraviolet absorption value data of the spectrometer 32, and calculates the protein concentration and the total amount of target protein flowing through the flow cell 10 per unit time in real time according to a preset calibrated standard curve; The total amount of the target protein is sent to the protein load control device 40; preferably, the protein concentration calculation device 33 is an industrial computer.
  • the flow cell 10, the ultraviolet irradiation device 31 and the spectrometer 32 in the protein concentration detection and calculation device 30, and the connection between them are the in-situ UV absorbance detectors of the present invention.
  • the detection time of the on-site UV absorbance detector of the present invention is 10 microseconds to 10 seconds. It is arranged at the entrance of the convergence layer (for example, the main unit of cation exchange chromatography/the main unit of anion exchange chromatography).
  • the protein concentration calculation device 33 When the chromatography device 20 is running, the protein concentration calculation device 33 will calculate the accumulated sample loading amount in real time based on the real-time measured concentration, the sample flow rate of the chromatography device 21, and the sample loading time, and transmit the data to the DCS system in real time .
  • the DCS system compares this data with the pre-set sample loading capacity of the chromatography column 22. If the two are equal, the DCS system will control the chromatography device 21 to stop loading the sample and enter the subsequent purification step. This prevents the loading of the sample from exceeding the loading capacity of the chromatography column 22.
  • the ultraviolet spectrometer 32 is zeroed for concentration measurement.
  • the solution containing the target protein flowing from the chromatography device 20 passes through the flow cell 10, an ultraviolet absorption peak is generated.
  • the relationship between the absorption intensity and the concentration of the solution containing the target protein conforms to Lambert Beer's law within a certain range, which can be calculated by Formula 1. .
  • c is the monoclonal antibody concentration
  • A is the UV absorbance value
  • £ is the extinction coefficient
  • L is the 10 optical path of the flow cell.
  • the monoclonal antibody concentration c can be calculated based on the UV absorption value A.
  • the ultraviolet absorption value data of the spectrometer 32 is transmitted to the industrial computer, and the industrial computer calculates the monoclonal antibody concentration in real time according to the pre-calibrated standard curve.
  • the total amount of mAb flowing through the flow cell 10 per unit time can be calculated by Equation 2:
  • V is the flow rate of the solution containing the target protein
  • m is the total amount of monoclonal antibody
  • t is the time.
  • the flow rate of the solution containing the target protein is obtained by the real-time flow rate of the chromatography device 21, and the time integral operation is calculated in real time by the industrial computer.
  • the total amount of monoclonal antibody calculated in real time is sent by the industrial computer to the DCS system for controlling the loading capacity of the chromatography device 21.
  • the industrial computer is sent to DCS (Distributed Control System).
  • the communication protocol between the systems adopts the industrial communication protocol (ModBusRTU).
  • the OPC communication protocol is adopted between the chromatography device 20 and the protein concentration detection and calculation device 30.
  • HPLC online detection is mainly used in the cation purification step.
  • the existing process adopts offline detection of charge isomers, which takes 20-24 hours, and it is necessary to wait for the detection result to determine the sampling interval of the purification step, which reduces the production efficiency.
  • the waiting time is greatly shortened or eliminated, and the efficiency is improved.
  • Embodiment 2 Affinity Chromatography Main Unit
  • GE is selected as the chromatography device Multi-column purification system ( PCC), which includes three chromatography columns. Each chromatographic column has a UV detector before and after it, and judges whether the chromatographic column has reached the preset end point based on the UV signal of the injection liquid of the specific chromatographic column and the UV signal of the effluent.
  • the PCC system can only detect the change in the concentration of the feed liquid and estimate the loading state of the packing material based on this.
  • Management system according to The PCC system contains a program that issues instructions to actuators (such as pumps or valves) to push the flow of material to flow or stop in the flow path.
  • actuators such as pumps or valves
  • the virus removal main unit 1 is a low pH virus inactivation system, which mainly includes a low pH virus inactivation tank (virus removal main unit 1 (Tank2)), a pH sensor installed on the tank, and respectively connected to the acid tank and the alkali tank Adding acid pump and adding alkali pump.
  • Virus removal main unit 1 (Tank2) and buffer volume 2 (Tank1) are connected through a pump, and can automatically adjust the pH value of the affinity chromatography collection liquid through an acid/base pump according to the program setting. After the virus inactivation is completed, the neutralization is adjusted back to the set pH value, and the material liquid flows out from the virus removal main unit 1 (Tank 2) to the buffer volume 3 (Tank 3) after deep filtration.
  • the DCS system judges whether the conductivity value of the buffer volume 3 (Tank3) is within the set target range. If it is not within the range, conduct conductivity adjustment.
  • the chromatography device can be a multi-column purification system.
  • the chromatography device includes a chromatography column.
  • the entrance of the chromatography column is equipped with the in-situ UV absorbance detector of the present invention.
  • the management system issues instructions to actuators (such as pumps or valves) according to the operation status of adjacent steps, the combination state of ion exchange chromatography media and the preset algorithm to push the flow of material to flow or stop in the flow path.
  • the in-situ UV absorbance detector is arranged at the entrance of the cation exchange chromatography column.
  • the solution flow channel interfaces (11, 12) of the flow cell 10 include a solution inflow interface 11 and a solution outflow interface 12 arranged at both lateral ends of the flow cell;
  • the optical channel interface (13, 14) It includes an upper end interface 13 and a lower end interface 14 arranged at both longitudinal ends of the flow cell, and in the middle of the flow cell 10 there is an optical aperture 15 for the solution containing the target protein to pass;
  • the flow The cell 10 is a variable optical path flow cell; the optical path of the variable optical path flow cell is realized by adjusting the size of the optical aperture 15, and the adjustment can be adjusted by a vernier caliper; specifically, the optical aperture 15 It is constituted by the distance between two optical fibers 151 arranged in the middle of the flow cell 10, and the distance between the two optical fibers 151 is 0.1 mm to 10 mm.
  • Adjusting the distance between the two optical fibers 151 can change the optical path length of the variable optical path flow cell 10, and changing the optical path length of the flow cell 10 is to adjust the range of the measurable monoclonal antibody concentration of the instrument.
  • the concentration range is between 100g/L and 0.1g/L.
  • the protein concentration detection and calculation device 30 includes: the solution inflow port 11 of the flow cell 10 is connected to the chromatography device 21, and the solution outflow port 12 of the flow cell 10 is connected to the chromatography column 22; the upper port 13 It is connected to the ultraviolet irradiation device 31, and the lower port 14 is connected to the spectrometer 32.
  • the chromatography device 21 provides a sample stream (that is, the solution stream containing the target protein). After the sample passes through the flow cell 10, it flows out from the solution outflow port 12, and the solution outflow port 12 is connected to the chromatography column 22.
  • the upper interface 13 is an optical fiber interface, and is connected to the light source through an optical fiber 151.
  • the lower end interface 14 is an optical fiber interface, and is connected to the spectrometer 32 through an optical fiber 151.
  • the light emitted by the light source of the ultraviolet irradiation device 31 is absorbed by the monoclonal antibody flowing between the two optical fibers 151 in the flow cell 10, and the spectrometer 32 detects the transmitted light.
  • the concentration of the monoclonal antibody sample is directly proportional to the UV absorption intensity, so the monoclonal antibody concentration can be calculated.
  • the optical fiber is in the flow path, since the material liquid flowing through the slit of the optical fiber is small, most of the material liquid passes through the space around the light without causing flow resistance.
  • the two optical fibers 151 are connected to the flow cell 10 through a threaded interface, and the optical fiber 151 connected to the upper end interface 13 is connected to the light-emitting light source of the ultraviolet irradiation device 31; the light-emitting light source preferably adopts Heraeus FiberLight (deuterium Lamp), the deuterium lamp emits more stable.
  • the optical fiber 151 connected to the solution outflow interface 12 is connected to the spectrometer 32.
  • the optical fiber 151 is an anti-ultraviolet quartz optical fiber, and the core diameter of the optical fiber 151 is 450 micrometers; the two optical fibers 151 are connected to the flow cell 10 through SMA905 joints.
  • the flow cell 10 is made of stainless steel, and the two optical fibers 151 are connected to the flow cell 10 with threaded interfaces (for example, SMA905).
  • One optical fiber 151 connected to the upper port 13 is connected to a light source for providing ultraviolet light of a specific wavelength (for example, 280 nm), and the other optical fiber 151 connected to the lower port 14 is connected to a spectrometer 32 to absorb the light from the monoclonal antibody.
  • the signal is transmitted to the spectrometer 32 to obtain the absorbance value A.
  • Spectrometer 32 uses OCEAN-FX-UV-VIS-ES single-wavelength spectrometer, which can manually set the wavelength (200nm-850nm); the detection time of spectrometer 32 is 10 microseconds-10 seconds, and the detection range of spectrometer 32 is 0-2AU
  • the optical path of the flow cell 10 can be adjusted by changing the distance between the two optical fibers 151, usually between 0.1 mm and 10 mm.
  • the measurable monoclonal antibody concentration is between 100g/L and 0.1g/L.
  • the luminescent light source is a light source machine, and the light source machine is an ultraviolet irradiation device 31, and the ultraviolet irradiation device 31 irradiates the protein solution between the two optical fibers 151 of the flow cell 10.
  • the light sources that can be selected include but are not limited to xenon lamps, deuterium lamps, LED lamps, mercury lamps, etc.
  • the wavelength range of the emitted light should include 200nm-400nm, preferably the wavelength is between 270nm-320nm.
  • the spectrometer 32 that can be selected includes, but is not limited to, an ultraviolet spectrometer, an ultraviolet-visible spectrometer, etc., and the absorbance value range is usually between 0-2 AU.
  • the available absorbance value range is usually 0.05-1.6 AU.
  • Ordinary silica fiber will form a defect center in the fiber under the irradiation of strong ultraviolet light (below 300nm), resulting in a decrease in fiber transmittance.
  • the optical fiber is an anti-ultraviolet silica fiber with a core diameter of 450 microns, and the material is anti-ultraviolet radiation silica XSR190-1100nm.
  • the two optical fibers are connected to the flow cell 10 through a connector SMA905.
  • the online target protein quantitative detection system 100 performs quantitative detection of target protein, specifically:
  • Flow cell 10 stainless steel flow cell, variable optical length (the vernier caliper is manually adjusted to about 0.5mm, which is the distance between the two optical fibers 151); sample: Mab1
  • the online target protein quantitative detection system 100 determines the cumulative amount of protein per unit time
  • Flow cell 10 stainless steel flow cell, variable optical path (the vernier caliper is manually adjusted to about 0.15mm, which is the distance between the two optical fibers 151); sample: Mab2
  • the online target protein quantitative detection system 100 determines the cumulative amount of protein per unit time
  • the online target protein quantitative detection system 100 measures and measures the cumulative amount of protein within a unit time, and the measurement speed is fast, which can reach 1 ms at the fastest. Other programs are 1s or more.
  • the measurable range is wide, and a single flow cell 10 can measure the concentration range of 0.1g/L to 100g/L. Data communication can be carried out with the DCS system; the amount of monoclonal antibody flowing through the flow cell 10 within a certain period of time can be calculated.
  • the injection loop is connected to the six-way valve position No. 2 and No. 5
  • the HPLC pump is connected to the six-way valve position No. 1
  • the HPLC analytical column is connected to the six-way valve position No. 6.
  • the No. 4 position of the through valve is connected to the flow path of the purification system through the sampling pipeline.
  • the online HPLC detection method is:
  • Sampling (Bypass/Loop loading): DCS sends instructions to switch the six-way valve, and the sample flows into the six-way valve from position 4 and into the injection loop through position 5. At a certain flow rate, after the preset sample loading time, the sample loading process of the sample loop is completed, and the DCS sends an instruction to switch the six-way valve and enter (2) the sample analysis step.
  • the detector collects the detection signals at different time points, transmits them to the Matlab software, and calculates the purity of the target molecule to determine whether the sample meets the sample standard. If the calculated sample purity data is less than the product purity setting value, the corresponding fraction is converted into waste liquid. If the sample purity is greater than or equal to the product purity setting value, the corresponding fraction is collected in the product collection tank (ie, the downstream buffer volume of the chromatography main unit in this step, such as the buffer volume 4 (Tank 4) shown in Example 1). Due to the realization of merging fractions based on real-time measurement of product purity, the variability of product quality is minimized.
  • the online target protein quantitative detection system enters the standby state. After the equilibration step is performed for about 20 minutes, make sure that the UV flow cell has been flushed, and zero the online target protein quantitative detection system.
  • the DCS system sends a start protein accumulation instruction to the online target protein quantitative detection system, starts to calculate the sample protein amount in real time, and sends the calculation result to the DCS system.
  • the DCS system sends an instruction to stop loading
  • the DCS system will send an instruction to stop sample loading and start the next step.
  • the HPLC method is a self-developed fast CEX-HPLC method with a detection time of only 4.7 minutes.
  • the cation elution retention time is 6 minutes, so each elution volume (column volume, CV) can be detected.
  • HPLC analysis results are transferred to Matlab software.
  • the self-developed program will perform the sample combination calculation according to the given sample combination standard (for example, acidic isomer peak ⁇ 30%, main peak>60%, Basic isomer peak ⁇ 15%).
  • the program and the DCS system are connected through a data interface included in the program.
  • Those skilled in the art can replace the self-developed program used in this step with commercially available workstations, program packages or applications according to actual conditions.
  • the chromatography device can be a multi-column purification system.
  • the chromatography device includes a chromatography column.
  • the entrance of the chromatography column is equipped with the in-situ UV absorbance detector of the present invention.
  • the management system issues instructions to actuators (such as pumps or valves) according to the operation status of adjacent steps, the combination state of ion exchange chromatography media and the preset algorithm to push the flow of material to flow or stop in the flow path.
  • the virus removal main unit 2 is a virus removal filtration system (nanofiltration), including a constant pressure pump and a virus removal filter.
  • a constant pressure pump is connected to the buffer volume 5 (Tank5), and the flow rate of the diaphragm pump is controlled to be constant at the setting.
  • Virus removal filtration is performed under pressure (29 psi), and the sample flows out of the virus removal main unit 2 after filtration.
  • Concentration and/or liquid exchange main unit 1 (one-way tangential flow filtration (SPTFF))
  • the concentration and/or liquid exchange main unit 1 is an ultrafiltration concentration system, which is composed of a constant pressure pump and a unidirectional tangential flow concentration membrane package. One inlet of the constant pressure pump is connected to the buffer volume 6 (Tank6). The concentration is carried out at 1.5Bar), and the sample flows out of the main unit 1 for concentration and/or liquid exchange after concentration.
  • the membrane pack is rinsed with a predetermined rinse volume.
  • Concentration and/or liquid exchange main unit 2 (tangential flow liquid exchange)
  • the concentration and/or liquid exchange main unit 2 is an ultrafiltration liquid exchange system, which is composed of a pump and a one-way in-situ washing filter membrane package.
  • the pump inlet is connected to buffer volume 7 (Tank7), and one-way washing is performed under the conditions set by the program. filter.
  • the peristaltic pump is used as an actuator to adjust the flow or stop of the material flow. Control the pressure at the inlet of the tangential flow exchange unit to be constant pressure (15-25psi). The speed ratio of the feed peristaltic pump and the replacement fluid peristaltic pump is adjusted according to a constant ratio (about 4-5 times).
  • the sample is the final product solution.
  • this embodiment is provided with a purification system including a convergent layer.
  • the main unit of affinity chromatography includes 7 subunits, which are realized by operating three chromatography columns in a bind-elution mode for 2 or 3 cycles, respectively.
  • the affinity chromatography main unit includes 7 sub-units, its output stream includes 7 sub-streams.
  • the main unit of cation exchange chromatography includes 3 subunits, which are realized by running a chromatography column in bind-elution mode for 3 cycles; or includes 1 subunit, which passes through a chromatography column in bind-elution mode Run 1 cycle to achieve.
  • the anion exchange chromatography main unit includes 1 subunit, which is realized by running a chromatography column in flow-through mode for 1 cycle.
  • the main unit of cation exchange chromatography includes 1 subunit
  • each affinity chromatography slave unit is usually independently tested by the sample detection system. Therefore, the unit operation of the affinity chromatography master unit requires the sample detection system to run 7 times.
  • the main unit of cation exchange chromatography is only equipped with a set of detectors. Accordingly, the unit only needs to run the sample detection system once in a running cycle.
  • FIG. 10 A schematic diagram of a purification system with two convergent layers (the main unit of cation exchange chromatography and the main unit of anion exchange chromatography as the convergent layer) is shown in FIG. 10. in:
  • the main unit of cation exchange chromatography includes 3 subunits

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Abstract

一种用于进行连续纯化的纯化系统和方法，纯化系统包含：(1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；(2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个病毒灭活亚单元；(3)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元；在每个主单元的上游或下游各自独立地任选存在的一个或更多个缓冲体积；每个主单元及缓冲体积之间通过可使料液流通的管路连接，其中，每个缓冲体积配置至少一个重量传感器和/或至少一个液位传感器，重量传感器和液位传感器与管理系统通信连接；管理系统根据重量传感器和/或液位传感器的信号对纯化系统进行启动、继续和停止的控制；纯化系统纯化的靶分子是蛋白；浓缩和/或换液单元是用于实施以连续操作进行的超滤或透析的单元，在浓缩和/或换液单元中，包含样品的流股的流动方向与滤出方向不同。

Description

连续纯化生物制药产品的系统和方法
本申请要求2020年03月30日提交的中国专利申请202020432845.9和2020年12月02日提交的202011400115.1的优先权，其全部内容通过引用整体并入本申请。
技术领域
本发明涉及生物制药领域，并且具体地涉及一种连续流纯化工艺，纯化生物制药产品的系统。
背景技术
生物制药的靶分子通常为大分子，其生产过程较为复杂，包含多步纯化步骤及严格的清洁灭菌、除病毒流程。然而由于生物产品生产的复杂性，并且，为满足制药产品生产技术要求和监管规范，需要在生产过程中对多项指标进行检测和控制，不利于药品生产的连续化纯化生产，导致生产效率不高，产能不足，产品价格昂贵。
WO2020148119A1公开了将单克隆抗体的分批纯化工艺转化为连续纯化工艺的方法。其中抗体下游过程采用蛋白A(Protein A)亲和层析、阳离子交换层析、阴离子交换层析、病毒过滤和超滤/透析。Protein A亲和层析使用具有5根色谱柱的Bio SMB色谱法，可以生成连续的进料流和连续的洗脱液流。CEX采用并行批处理模式(parallel batch mode)，病毒过滤和超滤/透析均采用批处理模式(batch mode)。
CN106794424A公开了层析处理单元等与切向流过滤处理单元流体连通的连接系统使用的控制系统和方法。
发明内容
本发明提供一种进行连续纯化的纯化系统，其用于靶分子的连续、封闭和自动化纯化。
在一方面，本发明提供一种用于进行连续纯化的纯化系统，其包含
(1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；
(2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个病毒灭活亚单元；和
(3)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元；
在每个主单元的上游或下游各自独立地任选存在的一个或更多个缓冲体积；每个所 述主单元及缓冲体积之间通过可使料液流通的管路连接，
其中，每个缓冲体积配置至少一个重量传感器和/或至少一个液位传感器，所述重量传感器和液位传感器与管理系统通信连接；
所述管理系统根据所述重量传感器和/或液位传感器的信号对所述纯化系统进行启动、继续和停止的控制；
所述纯化系统纯化的靶分子是蛋白；
所述浓缩和/或换液单元是用于实施以连续操作进行的超滤或透析的单元，在所述浓缩和/或换液单元中，包含样品的流股的流动方向与滤出方向不同。
在另一方面，本发明提供一种纯化靶分子的方法，所述方法通过本发明的系统完成。
附图说明
图1为包含一个汇聚层的操作层示意图。
图2为单抗纯化系统示意图。
图3为在线目标蛋白定量检测系统组成示意图。
图4为在线目标蛋白定量检测系统组成示意图。
图5示出在线目标蛋白定量检测系统中流通池与系统其他装置的连接。
图6为在线目标蛋白定量检测系统中的流通池的结构示意图。
图7为图6中的A处放大图。
图8为在线HPLC装置示意图。
图9为存在一个汇聚层(阳离子交换层析主单元作为汇聚层)的纯化系统示意图。
图10为存在二个汇聚层(阳离子交换层析主单元作为汇聚层)的纯化系统示意图。
具体实施方式
以下将对本发明进一步详细说明，应理解，所述用语旨在描述目的，而非限制本发明。
一般术语和定义
除非另有说明，本文使用的所述技术和科学术语具有与本发明所属领域技术人员通常所理解的相同的含义。若存在矛盾，则以本申请提供的定义为准。当以范围、优选范围、或者优选的数值上限以及优选的数值下限的形式表述某个量、浓度或其他值或参数的时候，应当理解相当于具体揭示了通过将任意一对范围上限或优选数值与任意范围下限或优选数值结合起来的任何范围，而不考虑该范围是否具体揭示。除非另有说明，本文所列出的数值范围旨在包括范围的端点和该范围内的所有整数和分数(小数)。
除非文中另有说明，单数形式指代如“一种”、“该”，包含复数指代。表述“一种(个)或多种(个)”或者“至少一种(个)”可以表示1、2、3、4、5、6、7、8、9种(个)或更多种(个)。
术语“约”、“大约”当与数值变量并用时，通常指该变量的数值和该变量的所有数值 在实验误差内(例如对于平均值95％的置信区间内)或在指定数值的±10％内，或更宽范围内。
术语“计量比”是将各种物质按一定的重量进行配比。
术语“任选”或“任选存在”是指随后描述的事件或情况可能发生或可能不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。
表述“包含”或与其同义的类似表述“包括”、“含有”和“具有”等是开放性的，不排除额外的未列举的元素、步骤或成分。表述“由…组成”排除未指明的任何元素、步骤或成分。表述“基本上由…组成”指范围限制在指定的元素、步骤或成分，加上任选存在的不会实质上影响所要求保护的主题的基本和新的特征的元素、步骤或成分。应当理解，表述“包含”涵盖表述“基本上由…组成”和“由…组成”。
在本文中可交换使用的术语“加工步骤”或“单元操作”指在纯化过程中使用一种或多种方法或装置以实现某种结果。可以用于纯化过程的加工步骤或单元操作的实例包括但不限于澄清、层析、病毒灭活、以及浓缩和/或换液。应当理解每个加工步骤或单元操作可以采用一个以上的步骤或方法或装置以实现该加工步骤或单元操作的预期结果。
术语“物料转换”广义地定义为特定物料在物理、化学或生物条件的影响下转换为另一种具有不同特性的物料。这种转换可以体现为物理性质的变化(例如熔化，溶解等)、化学性质的变化(例如化学反应，这样的反应可以是生物条件下进行的，例如酶催化的反应)或者作为混合物的物料在组成上发生变化(例如纯化过程)。其中，“物料”是指生产加工的对象，以及生产加工的产品。笼统地说，“物料”是指生产过程作用的物质对象，其涵盖的时间跨度为由生产过程初始(原料)至生产过程结束(最终产品)。
术语“批次生产”或“批次制造”表示相同的意义，并在本文中可以互换使用。批次生产是指以“批次工艺(batch process)”(也称为“批次处理(batch process)”、“间歇处理(batch process)”)或“批次操作(batch operation)”为特征的生产。
术语“连续生产”或“连续制造”或“连续纯化”(continuous manufacture,CM，或continuous purification)是指以连续工艺(continuous process)(也称为“连续处理(continuous process)”)或连续操作(continuous operation)为特征的生产或纯化。其中“连续”是指连续地将原料输入反应器，反应产物也连续地由反应器输出。在每一个时间点都同时有作为原料输入和作为产物输出的物料移动。“连续生产”或“连续制造”或“连续纯化”实现连续、封闭和自动化的进行靶分子的分离纯化，包括至少两个CM单元操作运行在时间上至少有一部分重叠。
连续制造可以在例如以下方面改进药品制造。例如，使用集成工艺，步骤减少，处理时间缩短；需要的设备占地面积较小；支持增强开发方法(例如，设计质量(QbD)和使用过程分析技术、使用数学模型)；对产品质量实时监控；并提供灵活的操作使得可以方便地进行工艺放大(scale-up)、工艺缩小(scale-down)和工艺外扩(scale-out)，以适应变化的供应需求。因此，采用连续制造进行药品生产被期望减少药品质量问题，降低制造 成本，提高患者获得优质药物的机会。Novasep(法国Pompey)，Tarpon(马萨诸塞州伍斯特)，Semba(威斯康星州麦迪逊)和GE Healthcare(新泽西州皮斯卡塔韦)提供了几种商业化的连续层析系统，例如GE Healthcare的
Figure PCTCN2021083516-appb-000001
PCC系统。Warikoo等人报道了
Figure PCTCN2021083516-appb-000002
PCC系统与上游连续生物反应器整合，形成用亲和层析纯化单抗或重组酶的连续生物处理平台。
“仓对仓(bin to bin)”方式的连续制造是指通过在单个、断开的CM单元操作之间持续地进行物料转移，在工艺设计的较高维度(at a higher level of granularity)实现工艺链的连续操作。
本发明的连续制造方法指纯化靶分子的方法，其包括两个或更多个加工步骤(或单元操作)，从而来自一个加工步骤的产出直接流入方法中的下一加工步骤，没有中断和/或不需要在进行下一加工步骤之前收集来自加工步骤的整个体积的产出。在一优选实施方案中，两个或更多个加工步骤可以在它们的至少一部分持续时间中同时进行。换句话说，在连续方法的情况下，如本文所述，不必在下一加工步骤开始之前完成加工步骤，但是一部分样品移动通过加工步骤。术语“连续”还用于加工操作内的步骤，在这种情况下，在包括多个步骤的加工操作进行期间，样品连续流过进行加工操作所需的多个步骤。本文所述的这样的加工操作的一个实例是亲和层析步骤与浓缩和/或换液步骤。
本文所述的这样的加工操作的一个实例是抛光纯化操作，其包括以连续方式进行的多个步骤。在一特别具体的实例中，这样的加工操作包括CEX层析、阴离子交换/阳离子交换混合介质处理、AEX层析中的两个或更多个。
本文所述浓缩和/或换液步骤是指减少流体物料中的液体体积和/或交换缓冲体系。该步骤具体的作用是浓缩、换液或者浓缩和换液，可以由本领域技术人员根据步骤本身特性和/或工艺流程中与所述浓缩和/或换液步骤相邻的步骤的特性来判断。浓缩和/或换液步骤可以采取的方法例如有过滤和透析。过滤可以采用例如膜法(例如超滤)或凝胶过滤层析。过滤可以是切向流过滤(tangential flow filtration或cross flow filtration)或死端过滤(normal flow filtration或dead-end filtration)，优选地为切向流过滤，特别是单向切向流过滤(single-pass tangential flow filtration,SPTFF)。透析优选地为在位透析(in-line diafiltration,ILDF)。
如本文所述，连续方法还包括这样的方法，其中任何单一的单元操作中流体物质的输入或输出是不连续或间断的。这样的方法也可以称作“半连续”方法。例如，在本发明的一些实施方案中，单元操作(例如，结合和洗脱层析步骤)中的输入可以连续装载；但是输出可以间歇地收集，其中纯化过程中的其他单元操作是连续的。因此，在一些实施方案中，本文所述的方法和系统包括至少一个以批次操作进行的单元操作，而方法或系统中的其他单元操作可以以连续方式操作。
本文中的“缓冲体积”的作用在于实现连续工艺中物料分布的调节，提供缓冲以使物料以期望的流速在单元操作之间流动。缓冲体积可以是任何已知的形式，例如容器(比 如缓冲罐、储液袋)，或可膨胀的管。应当理解，所述容器可以具备完全固定的形状，也可以是全部或部分可以发生形变的。
术语“缓冲罐”指在加工步骤之间或加工步骤内(例如，当单一的加工操作包括一个以上的步骤时)使用的任何容器或器皿或袋；其中来自一个步骤的产出流过缓冲罐至下一步。因此，缓冲罐不同于汇集罐，其并不意图盛放或收集来自步骤的整个体积的产出；而是取而代之使得来自一个步骤的产出能够连续流至下一步。
术语“单元”是指用于进行工艺技术步骤的装置或装置部分，或其以周期为单位的运行(operation cycle)。单元在纯化系统中能够实现特定功能。“单元”包括但不限于“主单元”和“亚单元”。“主单元”是指在一特定步骤中实现特定功能的装置或装置部分的集合，也可以理解为一个模块。主单元的实例包括但不限于层析主单元、浓缩和/或换液主单元、除病毒主单元，等。主单元之间通过流路相连接，以使物料在通过各个主单元的过程中，经历相应的工艺步骤。优选地，每个主单元以类似的补料速率同时运行，以便使生产时间最小化。“主单元”由一个或多个实现同类功能的“亚单元”组成。“亚单元”是指进行工艺技术步骤的装置或装置部分的一个周期的运行，也可以表述为一个运行周期。例如，任一亲和层析主单元可以包含一个或多个亲和层析亚单元，即一个或多个亲和层析柱各自独立地运行一个或多个周期。类似地，任一阳离子交换层析主单元、阴离子交换层析主单元或除病毒主单元可以包含一个或多个亚单元。“单元”的实施方式可以是已知能够实现期望的特定功能的装置。例如一个层析单元可以通过任何已知的层析装置实施，例如膜层析装置、层析柱，微珠、磁珠及其他固定相载体，多柱流动系统、流动床层析系统及其他层析系统，等。在一特定的实施方案中，一个层析亚单元是指一个层析柱或一个膜层析装置的一个运行周期，特别是以一个层析柱的一个运行周期。如本文使用的，单元具有一个或多个元件，元件选自工艺技术元件、服务技术元件、用于数据采集和处理的元件。
工艺技术元件(PTU,the process technology unit)包括用于进行步骤的元件，包括但不限于软管、过滤器、层析柱、容器等，其不与管理系统连接。
服务技术元件(the service technology unit,STU)包括单元的所有传感器和执行器。它们通过RIO与管理系统连接。STU的执行器可以是例如泵、阀，且传感器可以是例如UV测量、压力传感器或重量传感器等。
用于数据采集和处理的元件，在最简单的情况下是远程I/O、或局部智能，例如可编程逻辑控制(programmable logic control,PLC)或具有I/O层的基于PC的系统。在局部控制上执行单元的基本自动化。这两种系统变体在下文被称作RIO(registered input/output)。
术语“亚流”是指来自某一单元的向其相邻下游单元进行的物料输出形成的料流。其中，一个亚流是指某一亚单元向其相邻下游单元在时间上连续的一次物料输出形成的料流。例如，当亲和层析主单元为多柱位连续亲和层析设备时，其中任一层析柱的一个运 行周期为一亲和层析亚单元，每一亲和层析亚单元的输出流作为下一步骤(相邻下游单元)的一个输入流。当存在多个亲和层析亚单元时，亲和层析主单元向相邻下游单元的输出流包含了多个亚流。对于其他类型的主单元(例如阳离子交换层析主单元)的输出流，也应如此理解。两个亚单元的输出流之间相互连续是指其中第一个亚单元的输出流结束时，第二个亚单元的输出流正好开始，即，两个亚单元的输出流之间在时间上是连续的，并且可以认为两个输出流共同形成一个连续的流。两个以上亚单元的输出流之间相互连续的情况也应作类似理解。两个亚单元的操作时间之间相互连续是指其中第一个亚单元的操作结束时，第二个亚单元的操作正好开始，即，两个亚单元的操作之间在时间上是连续的。两个以上亚单元的操作时间之间相互连续的情况也应作类似理解。在流过模式的层析主单元包含多个亚单元的情况下，假如其中两个或以上亚单元的操作时间之间是连续的，则可以理解所述两个或以上亚单元的输出流之间也应当是相互连续的。应当理解，在通常的连续流工艺中，缓冲体积向相邻下游单元的输出是完全连续的，其在本文中描述为仅包含一个亚流的输出流。
一个运行周期对应一定量的样品输入及该样品输出的过程，并且样品输入和样品输出的时间可以部分重叠、完全重叠或完全不重叠。例如以结合-洗脱模式进行层析，其中一个运行周期包含一次(即，一定量的)样品输入(结合)-样品输出(洗脱)的过程，在一个层析柱的一个运行周期中，样品输入和样品输出的时间部分重叠或完全不重叠，优选地完全不重叠。在一些情况下，例如以流过模式进行层析时，样品输入的时间和样品输出的时间分别是完全连续的，此时可以将特定时间长度指定为一个运行周期，所述特定时间长度对应于特定的样品输入量和/或特定的样品输出量，并且所述特定的样品输入量和/或特定的样品输出量对应于紧邻上游单元一个运行周期输出的产品量。也就是说，当划分这样的装置或装置部分的运行周期时，可以根据需要，将该装置或装置部分用于处理紧邻上游单元一个运行周期产生的样品并作为产品向下游输出的时间划分为一个运行周期。其中，用于处理紧邻上游单元一个运行周期产生的样品的时间包括接受样品(上样)的时间，因此所述运行周期开始的时间为开始接受紧邻上游单元一个运行周期产生的样品的时间。当特定主单元的样品输入的时间和样品输出的时间分别是完全连续的，该主单元的一个运行周期可以定义为用于处理紧邻上游主单元一个运行周期产生的样品并作为产品向下游输出的时间。在一些情况下一段时间(例如一个运行周期)的终点与另一段时间(例如另一运行周期)的起点为同一时间点，这样的情况不属于时间段的重叠，这样的情况也包含在所述“完全不重叠”的情况中。
应当理解，为了实现连续生产，对于特定装置或装置部分，两个或更多个运行周期之间可以部分重叠、完全重叠或完全不重叠，优选地部分重叠或完全不重叠。例如在一些实施方案中，所述以流穿模式进行层析的一个层析柱的特定运行周期中用于处理紧邻上游单元一个运行周期产生的样品的时间可以与另一运行周期(例如，上一运行周期)的所述将产品向下游输出的时间部分重叠或完全重叠，重叠的程度可以根据实现连续生产 的需要来事先设定。在另一些实施方案中，所述以结合-洗脱模式进行层析的一个层析柱的特定运行周期与另一运行周期之间完全不重叠。为了实现连续生产，可以根据需要对特定主单元的多个亚单元的样品输入时间和/或样品输出时间进行统筹以实现该主单元样品输入时间的连续和/或样品输出时间的连续，或者，也可以根据需要借助缓冲体积，实现缓冲体积的紧邻上游主单元样品输出时间的连续和/或缓冲体积的紧邻下游主单元样品输入时间的连续。
应当理解，根据产品需求，或根据进样量，或根据纯化系统运行的时间长度，任一装置或装置部分可以任选地运行一个或多个运行周期，具体可以由管理系统进行控制。
在一实施方案中，任一进行工艺技术步骤的装置或装置部分(例如装置中的任一元件)的某个特定运行周期的长度均为可以测量、预期和控制的。
传统的抗体层析纯化步骤包括：(i)目标产物的浓缩和有害杂质的去除(捕获)，(ii)可选地，进一步去除大量杂质(enhance)，(iii)最后，去除剩余的痕量杂质和不需要的目标产物结构变体，例如目标产物的二聚体和多聚体(polish，“抛光”)。本文对于抗体层析纯化单元操作的讨论主要涉及亲和层析、离子交换层析，并且不排除任何已知可以用于纯化抗体的层析手段的使用，例如混合模式层析、疏水相互作用层析、尺寸排阻层析、羟基磷灰石层析，以及上述各种层析的任意组合。优选地，层析步骤选自亲和层析，离子交换层析及其组合。抗体的纯化还包括除病毒单元操作。除病毒单元操作可以采用已知的任意方法，只要其符合本发明的工艺设定的单元操作产品质量要求。除病毒单元操作的实例包括但不限于病毒灭活、除病毒过滤及其组合。
术语“工艺表征”是指对工艺进行表征的过程。其包括对参数进行测量、归类和评估。
术语“工艺设计空间”是指ICH Q8指南定义的设计空间。设计空间是指已证明可提供质量保证的输入变量(例如，材料属性)和过程参数的多维结合及相互作用。因此，设计空间由从过程表征研究中确定的重要和关键过程参数及其可接受范围来定义的。该定义本身不能由工艺设计者自行扩展，而是需要行业和监管机构来阐述。某种产品的设计空间必须经过监管评估和批准。建立设计空间的方法涉及工艺表征研究，并且总体上涉及三个关键步骤：1.进行风险分析以识别用于工艺表征的参数；2.使用DoE(design of experiments)试验设计来设计多变量的研究方案，以使研究能够获得适用于理解和定义设计空间的数据；3.执行研究方案并分析研究结果，以确定参数的重要性及其在建立设计空间中的作用，其中，评估参数对CQA的影响，基于统计学意义上显著影响CQA的工艺参数进行评价，并将确定对有CQA显著影响的工艺参数归类为CPP，并且计算重要和关键工艺参数的可接受范围，例如，通过估计的故障边缘的不确定性分析来确定参数的可接受范围。这些可接受的范围共同定义了设计空间。一般来说，可以通过放大/缩小数学模型来放大/缩小设计空间。
DoE一般用于查找仪器操作参数的范围，以了解样品制备的变化和方法精度的变化。
术语“表征范围”是指在工艺表征过程中检查的范围。“方法的可操作设计区间(method operable design range,MODR)”是指某一方法设计空间的中在监管允许的范围内可以改变的参数。
术语“质量源于设计(Quality by Design,QbD)”是指一种系统性的开发方法，它基于合理的科学和质量风险管理，从预定义的目标出发进行工艺设计，并强调对产品和过程的理解以及过程控制。QbD可促进制造工艺在批准的设计空间内的改进(例如使用PAT工具进行持续改进)，而无需进一步的法规审查，并可减少批准后的工艺变更申请。
术语“过程分析技术(Process Analytical Technologies,PAT)”被定义为：一个用于设计、分析、控制生产的体系，该体系通过实时测量原料、在制物料(in-process materials)及工艺的关键质量属性(critical quality attributes,CQA)和关键性能属性(critical performance attributes)，以保证最终产品质量(FDA PAT Guidance,2004)。PAT的概念是对设计空间的概念的补充。PAT的应用是QbD的一个组成部分，它基于对CQA的测量为工艺的反馈控制提供手段。
PAT框架的期望目标是设计和开发充分理解的工艺、在生产工艺结束时始终确保预设的质量。当达到以下情况时，认为工艺得到了充分理解：①识别和解释所有关键变化源；②变化可以通过工艺管理；③产品质量属性可以通过由所用原材料、工艺参数、生产、环境和其他条件建立的设计空间进行准确、可靠的预测。应用PAT可以获得对工艺的更根本性的了解，因此与传统生物制造相比有根本性的改进。例如，应用PAT可以帮助获得单变量或多变量统计过程控制(SPC或MSPC)模型。应当理解，仅仅通过改进分析技术并不能实现PAT，例如，对参数进行在线或在位检测并不意味实现PAT，因为其未识别和解释关键变化源，也没有为实现保证产品质量属性的在线控制提供帮助。QbD和PAT的预期目的仅为保证良好的产品质量，并且旨在开发可在更广泛的条件下运行的工艺。CQA的选择遵循同样的策略。然而采用PAT可能使CPP的选择和监测手段、控制策略相对于不采用PAT的情况发生变化，这样的变化是无法事先预期的。
术语“关键质量属性(CQA)”是一种物理、化学、生物学性质或特征，其应当在适当的限制，范围或分布内，以确保所需的产品质量。建立设计空间时，需要使现行生产质量控制程序始终如一地(有效地)达到关键质量属性范围。在设计空间内对工艺进行实施会让产品符合明确的CQA规范。
术语“关键工艺参数(critical process parameter,CPP)”是指工艺中显著影响CQA的工艺参数。
术语“检验结果偏差(out of specification,OOS)是指超标的检验结果，即，不符合法定质量标准或企业内控标准的检验结果。OOS是一种结果偏差，并且往往由生产操作引起的。为了工艺稳定性和确保产品质量，连续制造工艺一般需要预先考虑OOS的处理策略。OOS的处理一般包括执行相应的偏差分析(例如，是否是由于实验发生错误，如：实验不符合系统适应性或实验的接受标准(或不符合实验接受标准的一部份)，技术 /仪器、原料是否存在异常)和偏差处理流程。
在位(in-line)检测又称为原位(in-situ)检测。在线(on-line)检测技术通常需要修改生物反应器以转移样品流。离线(off-line)或旁线(at-line)检测属于不连续分析方法，涉及取样或者样品前处理。一些文献将“在位”与“在线”统称为在线。相应地，在一些实施方案中，本发明描述的“在位”检测、“在位”设备和“在线”检测、“在线”设备均可以以“在位(in-line)”或“在线(on-line)”的方式实施。在一些特定的实施方案中，本发明描述的“在位”检测或“在位”设备以“在位”的方式实施。
“上游”是指在纯化系统中处在物料流通过程的靠前或上游位置；“下游”是指在纯化系统中处在物料流通过程的靠后或下游位置，在整个纯化系统的物料流通过程中。某个主单元或缓冲体积所处的位置为上游还是下游是相对的，以病毒灭活主单元为例，在一些实施方案中，亲和层析主单元位于病毒灭活主单元的上游，同时，浓缩和/或换液主单元位于病毒灭活主单元的下游。
某单元的“紧邻单元”是指在某个单元与其紧邻的单元之间除连接管路之外，没有其他的功能单元存在，紧邻单元可以是主单元也可以是缓冲体积；某主单元的“紧邻主单元”指在某个主单元与其紧邻主单元之间除连接管路和缓冲体积之外，没有其他的主单元存在；“紧邻上游主单元”指在某个主单元的上游与该主单元紧邻的主单元。
“重量传感器”指可以直接或间接感受到“缓冲体积”或“缓冲罐”的重量的传感器；“液位传感器”指可以直接或间接感受到“缓冲体积”或“缓冲罐”内液面高度或位置的传感器。
本发明的系统
在一方面，本发明提供一种用于进行连续纯化的纯化系统，其包含：
(1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；
(2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个病毒灭活亚单元；和
(3)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元；
在每个主单元的上游或下游各自独立地任选存在的一个或更多个缓冲体积；每个所述主单元及缓冲体积之间通过可使料液流通的管路连接，
其中，每个缓冲体积配置至少一个重量传感器和/或至少一个液位传感器，所述重量传感器和液位传感器与管理系统通信连接；
所述管理系统根据所述重量传感器和/或液位传感器的信号对所述纯化系统进行启动、继续和停止的控制；
所述纯化系统纯化的靶分子是蛋白；
所述浓缩和/或主换液单元是用于实施以连续操作进行的超滤或透析的单元，在所述浓缩和/或换液主单元中，包含样品的流股的流动方向与滤出方向不同。
在一优选的实施方案中，所述纯化系统纯化的靶分子是蛋白，优选抗体，特别是单克隆抗体。在一实施方案中，所述亲和层析主单元依据靶分子的性质选自蛋白A亲和层析、蛋白G亲和层析、蛋白L亲和层析。在一具体的实施方案中，所述纯化系统纯化的靶分子是含有Fc片段的单抗。在另一具体的实施方案中，所述纯化系统纯化的靶分子是含有Fc片段的融合蛋白。
层析系统中的装置
1.概述
在一实施方案中，亲和层析主单元上游存在至少一个缓冲体积，并且任一主单元上游和下游均存在至少一个缓冲体积。
在一实施方案中，所述管理系统根据重量及液位信号来控制进行以下操作：
1.当亲和层析主单元上游的缓冲体积的重量传感器反馈值和/或液位传感器反馈值达到预设值时，纯化系统运行启动。在一优选的实施方案中，当亲和层析主单元上游的缓冲体积的重量传感器和液位传感器反馈值均达到预设值时，纯化系统运行启动。
2.当任一主单元上游的缓冲体积的重量传感器反馈值和/或液位传感器反馈值达到预设值时，启动该主单元运行。在一优选的实施方案中，当任一主单元上游的缓冲体积的重量传感器反馈值达到预设值时，启动该主单元运行。在另一优选的实施方案中，当任一主单元上游的缓冲体积的重量传感器和液位传感器反馈值均达到预设值时，启动该主单元运行。
3.当任一主单元上游的缓冲体积的重量传感器和/或液位传感器反馈值为下限值时，停止料液向该主单元转移，当单元操作完成后该主单元停止运行。在一优选的实施方案中，当任一主单元上游的缓冲体积的重量传感器和液位传感器反馈值均为下限值时，停止料液向该主单元转移，当单元操作完成后该主单元停止运行。在一实施方案中，当所述单元上游的缓冲体积的重量传感器反馈值再次到达2所述的预设值时，再次启动该主单元运行。
4.当任一主单元下游的缓冲体积的重量传感器和液位传感器反馈值均达到设定上限值时，该主单元停止运行。
在另一些方面，本发明还提供根据如前所述的用于进行连续纯化的纯化系统，其依次包含至少一个亲和层析主单元、至少一个病毒灭活主单元和至少一个浓缩和/或换液主单元；并且在至少一个所述亲和层析主单元的至少一个亲和层析亚单元目标料液流出所述亲和层析主单元之后，所述管理系统允许至少一个所述病毒灭活主单元的至少一个病毒灭活亚单元启动运行；在至少一个所述病毒灭活主单元的至少一个病毒灭活亚单元目标料液流出所述病毒灭活主单元之后，所述管理系统允许至少一个所述浓缩和/或换液主单元的至少一个浓缩和/或换液亚单元启动运行，并且所述管理系统允许所述亲和 层析主单元的第一亚单元目标料液流出所述亲和层析主单元之后，所述病毒灭活主单元与所述亲和层析主单元运行时间至少一部分重叠；所述管理系统允许所述病毒灭活主单元的第一亚单元目标料液流出所述病毒灭活主单元之后，所述病毒灭活主单元与所述浓缩和/或换液主单元运行时间至少一部分重叠。
应当理解，当描述一个以上的单元的运行时间至少一部分重叠时，表示所述单元的运行时间之间在时间轴上出现重叠，并且并不限定所述一个以上的单元的运行在同一时间点开始或在同一时间点结束。在一实施方案中，表述“运行时间至少一部分重叠”表示某一单元的运行时间的约1％、约5％、约10％、约15％、约20％、约25％、约30％、约35％、约40％、约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约95％或约100％重叠。
在另一些方面，本发明还提供根据如前所述的用于进行连续纯化的纯化系统，在亲和层析主单元和浓缩和/或换液主单元之间，进一步包括至少一个阳离子交换层析主单元和/或至少一个阴离子交换层析主单元，所述阳离子交换层析主单元各自独立地包含一个或更多个阳离子交换层析亚单元，所述阴离子交换层析主单元各自独立地包含一个或更多个阴离子交换层析亚单元。
在另一些方面，本发明还提供根据如前所述的用于进行连续纯化的纯化系统，其依次包含：
(1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；
(2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个病毒灭活亚单元；
(3)至少一个阳离子交换层析主单元，所述阳离子交换层析主单元各自独立地包含一个或更多个阳离子交换层析亚单元；
(4)至少一个阴离子交换层析主单元，所述阴离子交换层析主单元各自独立地包含一个或更多个阴离子交换层析亚单元；和
(5)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元。
在另一些方面，本发明还提供根据如前所述的用于进行连续纯化的纯化系统，在至少一个上游主单元的至少一个亚单元目标料液流出所述主单元之后，所述管理系统允许至少一个所述上游主单元的紧邻下游主单元的至少一个亚单元启动运行；并且所述管理系统允许任一所述上游主单元的第一亚单元目标料液流出所述上游主单元之后，所述上游主单元与其紧邻下游主单元运行时间至少一部分重叠。
在另一些方面，本发明还提供根据如前所述的用于进行连续纯化的纯化系统，所述连续纯化是连续、封闭、自动化的进行靶分子的分离纯化。
另外，本发明还提供一种用于靶分子的连续纯化方法，通过本发明的系统完成。其 在一实施方案中，所述方法还包括使包含靶分子的澄清的细胞收获液(CCCF)流入如前所述的至少一个亲和层析主单元上游紧邻的缓冲体积，并使所述CCCF经如前所述的纯化系统分离纯化的步骤。
在一实施方案中，所述纯化系统包括在线目标蛋白定量检测系统。在线目标蛋白定量检测系统包含在位UV吸光度检测器、蛋白质浓度计算设备和蛋白质载量控制设备。在位UV吸光度检测器检测物料的紫外吸收值并将数据传送到蛋白质浓度计算设备。蛋白质浓度计算设备实时计算所述蛋白质浓度以及单位时间流经的目标蛋白总量；并将所述目标蛋白总量发送至蛋白质载量控制设备(DCS)。在线目标蛋白定量检测系统可以实时判断层析主单元的上样量，并控制层析主单元的上样终点，从而控制上样载量。
在位UV吸光度检测器是在线目标蛋白定量检测系统的一部分。
在一实施方案中，在所述纯化系统流通管道的相对于至少一个层析单元的上游和/或下游的位置，配置有一个或多个在位UV吸光度检测器，其与管理系统通信连接；
管理系统根据所述在位UV吸光度检测器在位检测的信号，来控制所述层析单元的上样(加载样品的过程)终点。
在一实施方案中，所述在位UV吸光度检测器在位检测层析单元的进料信号，其代表向所述层析单元的入口提供的进料物料的成分，和/或
所述在位UV吸光度检测器在位检测层析单元的流出物信号，其代表所述层析单元流出物的成分。
在一实施方案中，纯化系统流通管道的相对于至少一个层析主单元的上游配置有一个或多个在位UV吸光度检测器。
在一实施方案中，在位UV吸光度检测器包括：流通池，紫外线照射装置和光谱仪。流通池包括溶液流通道接口和光通道接口。所述溶液流通道接口与层析装置连接。光通道接口包括设置于所述流通池与物料流向垂直的方向两端的光通道第一接口和光通道第二接口，其分别与紫外线照射装置和光谱仪连接。光谱仪检测所述紫外线照射装置照射流经所述流通池的所述含目标蛋白的溶液的紫外线强度，并将紫外吸收值数据传送到蛋白质浓度计算设备。
在一实施方案中，纯化系统中的至少一个层析主单元包含多个亚单元。在一特别的实施方案中，至少一个层析主单元中，采用多柱位层析系统来实现多个亚单元；所述多柱位层析系统中的每个层析柱各自独立地处于以下状态之一：a.加载步骤、b.非加载步骤和c.静息状态。
任选地，在所述多柱位层析系统中，在至少一个层析单元的上游和下游的位置分别配置有一个或多个在位UV吸光度检测器，其与管理系统通信连接。管理系统根据所述在位UV吸光度检测器在位检测的信号，控制层析单元在所述状态a、b和c之间切换。
所述加载步骤是指层析柱填料与期望发生亲和结合的样品进行接触；
所述非加载步骤包括：洗脱结合在层析柱填料上的样品，并任选地包括以下步骤中 的一个或多个：加载样品后洗涤非靶分子的杂质、洗脱样品后清洗层析柱、清洗层析柱后重新进行平衡；
所述静息状态是指没有样品与层析柱填料接触和/或层析柱与纯化系统流通管道的其他部分之间基本没有流体流通。
在一实施方案中，浓缩和/或换液步骤中不希望保留的液体或溶质在滤出方向上被去除。希望置换的液体或溶质在滤出方向上被加入到包含样品的流股(stream)中。包含样品的流股流向纯化系统流通管道的相对于浓缩和/或换液单元的下游方向。在一优选的实施方案中，所述浓缩和/或换液步骤为切向流过滤，特别是单向切向流过滤。在另一优选的实施方案中，所述浓缩和/或换液步骤为单向切向流过滤和/或在位透析。
在一实施方案中，所述管理系统包括用于数据处理的元件。
在一备选的实施方案中，纯化系统流通管道的相对于特定层析单元的上游和下游均配置有一个或多个在位UV吸光度检测器，管理系统通过如下方法控制层析单元的上样终点：管理系统使用所述进料信号和所述流出物信号来确定所述层析单元中的层析介质的结合状态，其包括使用所述进料信号和所述流出物信号来确定△信号和最大△信号，△信号是所述进料信号减去所述流出物信号，最大△信号是当所述流出物信号由于大致全部未结合组分已经经过所述层析单元中的层析介质这一事实而显示平稳期时所述进料信号减去所述流出物信号；
其中使用所述△信号来确定所述层析单元的穿透点和/或饱和点，所述穿透点和饱和点计算为所述最大△信号的相应的某一预定百分比；
达到穿透点和/或饱和点时，管理系统发出指令，结束上样操作。
在一实施方案中，管理系统根据层析单元中的层析介质的结合状态(例如所述柱是否达到穿透点和/或饱和点)和预先设置的算法发出指令，控制层析单元在所述状态a、b和c之间切换。
在另一实施方案中，所述纯化系统还包含一个或多个除病毒单元。在一具体的实施方案中，除病毒单元为除病毒过滤、病毒灭活或其组合。在一特别具体的实施方案中，病毒灭活的方式为低pH灭活。
在一实施方案中，所述除病毒单元配置有传感器，所述传感器与管理系统通信连接。在一具体的实施方案中，所述传感器选自pH传感器、电导传感器和料位传感器。
在一具体的实施方案中，所述缓冲体积是缓冲罐。
在一实施方案中，缓冲体积和/或除病毒单元还配置有其他传感器，优选地，所述其他传感器选自压力传感器、光学传感器及其组合。
在一实施方案中，管理系统还根据传感器的信号和预先设置的算法发出指令，控制物料在单元操作之间和/或在单元操作内部的流速。
在一实施方案中，特定主单元的运行启动后，根据管理系统的指令，物料由紧邻的上游缓冲体积或紧邻的上游主单元流入所述特定主单元，然后该主单元进行单元操作， 并产生输出流；输出流根据管理系统的指令流入下游紧邻的缓冲体积或紧邻的下游主单元；特定主单元的单元操作结束时，输出流的产生停止。
在一特别具体的实施方案中，所述纯化系统中的亲和层析主单元、除病毒主单元、浓缩和/或换液主单元在纯化系统的流通管路中由上游至下游分布。
在另一具体的实施方案中，所述纯化系统中的进料物料为使用宿主细胞表达重组蛋白后，得到CCCF。在一特别具体的实施方案中，所述CCCF是细胞培养后经过至少包括离心和深层过滤的步骤得到的。
在一备选的实施方案中，所述纯化系统还包含一个或多个额外的层析主单元，所述层析选自混合模式层析、疏水相互作用层析、尺寸排阻层析、羟基磷灰石层析，以及上述各种层析的任意组合。
2.在线目标蛋白定量检测系统
本发明的在位UV吸光度检测装置配置于纯化系统中，目的在于提供一种在线目标蛋白定量检测系统，以实现在目标蛋白纯化的过程中，对目标蛋白进行在线定量检测以及根据检测结果控制所述目标蛋白的上样载量。
本发明的在线目标蛋白定量检测系统，包括：
流通池，包括溶液流通道接口和光通道接口，所述溶液流通道接口与层析装置连接，所述光通道接口与所述蛋白浓度检测计算装置连接；
层析装置，将含目标蛋白的溶液接入所述在线目标蛋白定量检测系统，并对流经的含目标蛋白的溶液进行蛋白分离，获得目标蛋白溶液；
蛋白浓度检测计算装置，对所述含目标蛋白溶液的浓度进行检测，并实时计算所述含目标蛋白的溶液中的蛋白总量；
蛋白质载量控制设备，与所述蛋白浓度检测计算装置和所述层析装置通信连接，根据所述蛋白总量控制所述层析装置的上样载量。
在一实施方案中，所述层析装置包括：
层析设备，与所述流通池的溶液流通道接口连接，将所述含目标蛋白的溶液接入所述在线目标蛋白定量检测系统；
至少一个层析柱，与所述流通池的溶液流通道接口连接，对流经的所述含目标蛋白的溶液进行蛋白分离，将所述含目标蛋白的溶液中的其他成分去除，获得目标蛋白溶液。
在一实施方案中，所述蛋白浓度检测计算装置包括：紫外线照射装置，与所述流通池的光通道接口连接，照射流经所述流通池的所述含目标蛋白的溶液；
光谱仪，与所述流通池的光通道接口连接，检测所述紫外线照射装置照射流经所述流通池的所述含目标蛋白的溶液的紫外线强度，并将紫外吸收值数据传送到蛋白质浓度计算设备；
蛋白质浓度计算设备，接收所述光谱仪的紫外吸收值数据，并根据预设的标定的标准曲线，实时计算所述蛋白质浓度以及单位时间流经流通池的目标蛋白总量；并将所述 目标蛋白总量发送到所述蛋白质载量控制设备。
其中所述蛋白浓度检测计算装置中的蛋白质浓度计算设备和蛋白质载量控制设备被本发明的用于连续纯化的纯化系统中的管理系统包含。
其中所述流通池以及蛋白浓度检测计算装置中的紫外线照射装置和光谱仪及其之间的连接即本发明的纯化系统中的在位UV吸光度检测器。
在一实施方案中，所述蛋白质载量控制设备包括：
上样控制模块，将接收到的所述目标蛋白总量作为累计的上样量与预设的层析柱上样载量进行实时对比，并在所述累计的上样量与预设的所述层析柱上样载量相等时，控制所述层析设备停止上样；以及
选择调用模块，执行选择并调用所述预设的标定的标准曲线、UV调零信号、目标蛋白累计命令以及目标蛋白定量累计上限设定的控制操作。
在一实施方案中，所述蛋白质浓度计算设备为工控机；
在一实施方案中，所述蛋白质载量控制设备为DCS系统。
在一实施方案中，所述流通池的溶液流通道接口包括设置于所述流通池横向两端的溶液流入接口和溶液流出接口；所述光通道接口包括设置于所述流通池纵向两端的上端接口和下端接口，并且在所述流通池的中部具有供所述含目标蛋白的溶液通过的光径口；
所述流通池的溶液流入接口与所述层析设备连接，所述流通池的溶液流出接口与所述层析柱连接；所述上端接口与所述紫外线照射装置连接，所述下端接口与所述光谱仪连接。
在一实施方案中，所述的流通池为可变光程流通池；所述可变光程流通池的光程由调整所述光径口的大小调整；
所述光径口由所述流通池的中部设置的两根光纤的间距构成，所述两根光纤的间距为0.1mm至10mm。
在一实施方案中，所述上端接口和下端接口为光纤接口，所述两根光纤通过所述上端接口和下端接口与所述流通池连接，并且与所述上端接口连接的所述光纤与所述紫外线照射装置的发光光源连接；与所述下端接口连接的所述光纤与所述光谱仪连接。
在一实施方案中，所述光纤为抗紫外石英光纤，所述光纤的芯径为450微米；所述两根光纤通过SMA905接头与所述流通池连接。
在一实施方案中，所述紫外线照射装置的发光光源包括氙灯、氘灯、LED灯或者汞灯；所述紫外线照射装置的发光光源的发射光波长范围为200nm-400nm；
所述光谱仪包括：紫外光谱仪，紫外-可见光谱仪；所述光谱仪的吸光值范围为0-2AU。
在一实施方案中，所述紫外线照射装置的发射光波长范围为270nm-320nm；
所述光谱仪的吸光值范围为在0.05AU-1.6AU；
在一实施方案中，所述目标蛋白为单抗。
本发明的通过将在线目标蛋白定量检测系统设置包括：流通池，层析装置，蛋白浓度检测计算装置以及蛋白质载量控制设备。流通池包括溶液流通道接口和光通道接口，所述溶液流通道接口与层析装置连接，所述光通道接口与所述蛋白浓度检测计算装置连接；层析装置将含目标蛋白的溶液接入所述在线目标蛋白定量检测系统，并对流经的含目标蛋白的溶液进行蛋白分离，蛋白浓度检测计算装置对所述含目标蛋白的溶液的浓度进行检测，并实时计算所述含目标蛋白的溶液的蛋白总量；蛋白质载量控制设备与所述蛋白浓度检测计算装置通信连接，根据所述蛋白总量控制所述层析装置的上样载量，由此，实现了对目标蛋白进行在线定量检测以及根据检测结果控制所述目标蛋白的上样载量。此外，本发明采用的光谱仪性能更好，最短积分时间10微秒，响应更快，更加适合在线检测的需要，氘灯发光更稳定。本发明采用1个流通池实现了0.1-10mm的光程变化，结构更简单，可测量浓度范围更广。采用SMA-905接口为标准接口，适应性强；当料液通过流通池时，由于流过光纤狭缝中的料液很少，大部分从外部空间通过，不会造成阻力。多个装置间采用工业通信协议更稳定可靠；以及本发明提供更多在线控制功能。
3.在线HPLC装置
在线HPLC装置包含取样管路、样品环、六通阀、配置有泵和分析柱的HPLC，以及检测器，并且以泵推动样品在在线HPLC装置中的流动/停止。取样管路为在层析设备洗脱液出口管路上的支路，样品经取样管路进入六通阀中的样品环，然后又回到洗脱液出口管路中。在线HPLC装置向DCS发送信号并受DCS控制。当需要进样时，六通阀切换回路，由HPLC泵将样品环中的样品推入HPLC柱上。HPLC分析完成后，其结果传输至DCS系统中。当样品分析完成后，经数据处理和计算，然后根据结果由DCS系统切换相应的阀门，并根据合样标准指导合样。
在一实施方案中，所述在线HPLC装置的检测器为紫外可见吸收检测器，例如紫外吸收检测器或DAD检测器。
纯化系统中的层(level)
本发明的纯化系统的包括操作层和自动化层。
1.操作层
操作层包括所有工艺技术元件(PTU)和服务技术元件(STU)，例如所述多个层析主单元、任选存在的一个或多个浓缩和/或换液主单元、任选存在的一个或多个除病毒主单元、任选存在的一个或多个缓冲体积，以及单元中存在的软管、过滤器、层析柱、容器、传感器、泵、阀，等。
操作层包含按以下顺序连接的三个层：(1)原料输入层；(2)汇聚层；(3)产品输出 层。
原料输入层根据供应需求向系统输入待层析的原料。应当理解，由于生物分子的常见生产过程是基于涉及使用活生物体或培养细胞的过程，这些过程比化学合成更难控制，生产得到的物料的预期均质性相对较低，因此不同时间点向纯化系统输入的原料之间的差异性相对较高。在用于表征物料属性的参数组成的空间中，不同时间点向纯化系统输入的原料的属性分布于多点。
一个汇聚层是指具备以下I和II所述特点的一个层析主单元：
I.物料：
(1)向汇聚层输入的物料已经经过至少一个纯化步骤，例如层析步骤，因此在用于表征物料属性的参数组成的空间中，不同时间点向汇聚层输入的物料的属性分散的程度相对于原料而言已经降低，在一个或多个维度(即，一个或多个参数)上经过了汇聚；并且(2)根据预先设定的参数要求，汇聚层将物料在(1)的基础上进行进一步汇聚，使得由汇聚层输出的物料在预先设定的一个或多个维度(即，一个或多个参数)上具备比向汇聚层输入的物料更高的汇聚性。汇聚层输出的物料包含原料含有的基本全部靶分子或其一部分。
II.纯化系统：
在汇聚层和汇聚层下游的层析主单元中，需要配置的层析亚单元的数目呈现出与汇聚层上游相比减少的趋势。
以N表示某一层析主单元中包括的层析亚单元数目，则在一实施方案中，所述使层析亚单元数目减少的趋势可以用下式(1)和/或(2)表示：
(1)N(汇聚层)≤N(汇聚层上游相邻层析主单元)；
(2)N(汇聚层下游相邻纯化主单元)≤N(汇聚层上游相邻层析主单元)；
其中所述汇聚层下游相邻纯化主单元是层析主单元、除病毒主单元或浓缩和/或换液主单元。
本发明的纯化系统中，至少一个汇聚层是由PAT反馈控制的(详见下文“PAT用于反馈控制”)。
产品输出层根据供应需求由纯化系统输出产品。本发明的纯化系统输出的产品在一些情况下，优选在大部分情况下，更优选地基本在每次输出时能够满足产品质量要求。在一些实施方案中，所述产品质量要求是指技术要求和监管规范对于该阶段的产品(例如原液、半成品或成品)规定的要求。
在一实施方案中，操作层包含一个原料输入层、至少一个汇聚层和一个产品输出层。在一实施方案中，操作层包含一个原料输入层、一个或二个汇聚层和一个产品输出层。
物料在进入汇聚层之前，可以经过级分合并，物料流出汇聚层中的层析单元之后，还可以任选地对物料进行进一步的级分合并。所述级分合并是指将特定属性符合一定要求的样品合并在一起。在一实施方案中，汇聚层对物料的汇聚作用是通过层析纯化和随 后的级分合并来实现的。
汇聚层还起到调和步骤之间的工艺周期时间差异的作用。管理系统通过PAT，可以得知物料的分布情况，并由此确定整个工艺中一个或多个步骤的启动或停止，并最终实现生产时间最小化。因此，汇聚层还实现了操作时间的汇聚，即以下第III点：
III.汇聚层中的层析主单元包含一个层析亚单元；或者
汇聚层中的层析主单元包含多个层析亚单元；多个层析亚单元的操作时间相互
连续。
在一实施方案中，纯化系统包含依次连接的第一层析主单元、除病毒主单元、第二层析主单元、第三层析主单元和浓缩和/或换液主单元；第一层析主单元的输出流作为除病毒主单元的输入流，除病毒主单元的输出流作为第二层析主单元的输入流，第二层析主单元的输出流作为第三层析主单元的输入流；第三层析主单元的输出流作为浓缩和/或换液主单元的输入流。在一实施方案中，第一层析主单元包含7个亚单元，通过三根层析柱实现，除病毒主单元包含3个亚单元，通过一个低pH病毒灭活罐运行三个周期来实现，第二层析主单元包含3个亚单元，通过一根层析柱运行三个周期来实现，第三层析主单元包含1个亚单元。在一实施方案中，第一层析主单元的第1、第2亚单元操作完成后，除病毒主单元开始第一操作周期，第一层析主单元的第3、第4亚单元操作完成后，除病毒主单元开始第二操作周期，第一层析主单元的第5、第6、第7亚单元操作完成后，除病毒主单元开始第三操作周期。其中除病毒主单元的一个操作周期的操作时间短于第一层析主单元的两个亚单元的操作时间。在一实施方案中，第一层析主单元以结合-洗脱模式进行层析。在一实施方案中，除病毒主单元的第一操作周期完成后，第二层析主单元开始第一操作周期，除病毒主单元的第二操作周期完成后，经过等待时间t1，第二层析主单元开始第二操作周期，除病毒主单元的第三操作周期完成后，经过等待时间t2，第二层析主单元开始第三操作周期。在一实施方案中，第二层析主单元的第一、第二、第三操作周期在时间上完全连续，t1、t2分别等于第二层析主单元第二或第三操作周期开始的时间减去除病毒主单元第二或第三操作周期结束的时间。在一实施方案中，第一层析主单元为亲和层析主单元，第二、第三层析主单元分别为离子交换层析主单元。在一实施方案中，第二层析主单元以结合-洗脱模式进行层析。在一实施方案中，第三层析主单元以流过模式进行层析。
在一实施方案中，亲和层析主单元位于汇聚层上游。在一实施方案中，汇聚层包含阳离子交换层析主单元、阴离子交换层析主单元或其组合。在一实施方案中，浓缩和/或换液主单元位于汇聚层下游。
在一实施方案中，纯化系统依次包含：亲和层析主单元、阳离子交换层析主单元和阴离子交换层析主单元，其中阳离子交换层析主单元为汇聚层，汇聚层数量为1。以N表示某一层析主单元中包括的层析亚单元数目，则汇聚层的汇聚作用使层析亚单元数目减少的趋势可以用下式(1)和(2)表示：
(1)N(阳离子交换)≤N(亲和)；并且
(2)N(阴离子交换)≤N(亲和)。
在一实施方案中，纯化系统依次包含：亲和层析主单元、阳离子交换层析主单元和阴离子交换层析主单元，其中阳离子交换层析主单元为汇聚层1，阴离子交换层析主单元为汇聚层2，汇聚层数量为2。以N表示某一层析主单元中包括的层析亚单元数目，则汇聚层的汇聚作用使层析亚单元数目减少的趋势可以用下式(3)表示：
(3)N(阴离子交换)≤N(阳离子交换)≤N(亲和)。
应当理解，在整个工艺过程中，越接近产品输出层，物料中的靶分子富集程度上升，物料价值增加，稳健的质量控制显得越为重要。此时，简化的生产操作也有利于降低生产成本。尤其是考虑到在一些实施方式中，随着多个纯化步骤的完成，越接近产品输出层，物料的总量呈现出减少的趋势。因此在接近产品输出层的输出单元中，采用较为集中的生产方式，即，减少亚单元数目，至少在保证产品质量、降低生产成本这两方面是有利的。因此，汇聚层的存在以及PAT在汇聚层的应用是本申请工艺优化的一个重要方面。
2.自动化层
自动化层包括用于数据采集和处理的元件。在一实施方案中，自动化层是指RIO(即，工控机或具有I/O层的基于PC的系统，或其组合)。在一特定的实施方案中，工控机为可编程逻辑控制(PLC)。在一特别具体的实施方案中，自动化层是指具有I/O层的DCS系统。在另一特别具体的实施方案中，自动化层是工控机与具有I/O层的DCS系统的组合。
在一实施方案中，本申请纯化系统的管理系统是指工控机和DCS系统。在一实施方案中，DCS系统其采用多种通信协议与设备通信，其实例包括但不限于Profibus-DP、OPC、Modbus-RTU、4-20mA信号等。在一实施方案中，控制程序运行在DCS系统的主控制器或副控制器中。执行器或传感器产生的数据被DCS系统采集，并进行存储。
PAT用于反馈控制
在一实施方案中，本发明用于实现PAT的装置为本发明的在位UV吸光度检测器。在另一实施方案中，本发明用于实现PAT的装置为本发明的在位UV吸光度检测器与其他在位或在线检测技术的组合。在一实施方案中，所述其他在位或在线检测技术选自：在线或在位光谱方法(例如近红外(NIR)、中红外(MIR)或拉曼光谱，等)、在线或在位色谱方法(例如在线色谱法，比如在线HPLC)及其组合。在一特定的实施方案中，所述其他在位或在线检测技术为在线HPLC。
在一实施方案中，本发明的用于实现PAT的装置中，至少有一个配置于汇聚层。在一特别的实施方案中，管理系统根据用于实现PAT的装置获取的信号，控制以下操 作中的一个或多个：
D.来自上游单元的输出流或缓冲体积中的物料流出作为输入流流进汇聚层；
E.来自汇聚层包含的上游单元的输出流作为汇聚层包含的下游单元输入流流进所述下游单元；
F.来自汇聚层包含的上游单元的输出流作为汇聚层包含的下游单元输入流流进所述上游单元和所述下游单元之间的缓冲体积中；
G.由缓冲体积中流出，进入汇聚层包含的下游单元；
H.由汇聚层流出，进入下游单元，或进入上游单元和下游单元之间的缓冲体积。
在一实施方案中，包含一个汇聚层的操作层示意图如图1，其中操作D、E、F、G、H中的一个或多个由管理系统根据用于实现PAT的装置获取的信号来控制。
在一实施方案中，汇聚层配置有至少一个用于实现PAT的装置。在一具体的实施方案中，汇聚层配置有至少一个本发明的在位UV吸光度检测器。在另一实施方案中，汇聚层还配置有在线HPLC装置。在一具体的实施方案中，汇聚层包含至少一个层析单元，至少一个层析单元的上游配置有至少一个在位UV吸光度检测器，管理系统根据上文所述的方法来确定层析单元中的层析介质的载样量。在一实施方案中，根据层析亚单元中的层析介质的载样情况，确定是否需要在层析亚单元所属的层析主单元中增加新的层析亚单元，以装载样品、进行纯化，并且使得所述层析主单元的总运行时间跨度落在预先设定的范围内。
纯化系统的规模
纯化系统以小规模实施时，例如实验室规模实施时，可以使用例如微型设备、集成装置。以大规模实施时，例如工业生产制备时，可以使用大型层析塔、层析床等设备。
纯化系统的实例
在一特别具体的实施方案中，本发明的纯化系统依次包括缓冲体积1、亲和层析主单元、缓冲体积2、除病毒主单元1(除病毒罐)、缓冲体积3、阳离子交换层析主单元、缓冲体积4、阴离子交换层析主单元、缓冲体积5、除病毒主单元2、缓冲体积6、浓缩和/或换液主单元1、缓冲体积7、浓缩和/或换液主单元2。特别地，本发明的纯化系统如图2所示。
操作物料流入/流出缓冲体积的执行器为传输泵和各个缓冲罐、除病毒罐底部的罐底阀。
每个缓冲体积配置有重量传感器和液位传感器。除病毒罐配置有pH传感器、电导传感器、重量传感器和液位传感器。每个缓冲体积以及除病毒罐分别独立地设定一个料液下限和一个料液上限(用重量传感器检测值衡量或重量传感器和液位传感器检测值共同衡量)。以仅有重量传感器进行检测为例，当重量传感器的检测值达到或低于所述重 量下限时认为该缓冲体积不适宜再为下游主单元提供料液，从而停止料液向该主单元转移并在适当时停止下游主单元的运行；当重量传感器的值达到或高于所述重量上限时认为该缓冲体积不再适宜再接受上游主单元输入的料液，从而停止上游主单元的运行。
对于每个缓冲体积分别独立地设定一个料液检测区间，用于判断是否可以启动紧邻的下游主单元，该重量区间称为启动该缓冲体积的紧邻的下游主单元的区间，简称为该缓冲体积的启动区间。启动区间的下限即该缓冲体积的料液下限，启动区间的上限小于或等于该缓冲体积的料液上限。
纯化过程可以描述为如下步骤1-8：
1.作为原料的澄清的细胞收获液暂存于缓冲体积1中；
2.缓冲体积1以及缓冲体积2重量及液位控制亲和层析主单元：
当缓冲体积1重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积1流入亲和层析主单元；
由DCS系统启动PCC程序，运行亲和层析方法；
在亲和层析过程中，物料由亲和层析主单元流入缓冲体积2；
当缓冲体积1重量低于下限，由DCS系统向PCC发送Digital I/O信号，PCC收到信号后，结束上样，进入结束步骤。PCC程序结束后，亲和层析单元操作完成；
假如缓冲体积2重量达到设定的重量上限，由DCS控制PCC暂停运行，暂停至缓冲体积2重量重新处于启动区间内之后，PCC继续运行；
3.缓冲体积2、除病毒主单元1以及缓冲体积3重量及液位控制除病毒主单元1：
当缓冲体积2重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积2流入除病毒主单元1；
缓冲体积2料液转移至除病毒主单元1传输完成后，稳定1-2分钟，根据pH传感器数据，DCS系统判断除病毒主单元1的pH值是否在需要的范围内，然后根据需要由自动化控制的泵操作加酸或者加碱，进行低pH病毒灭活操作；
当缓冲体积2重量低于下限，结束物料向除病毒主单元1的转移；
除病毒主单元1的低pH病毒灭活操作完成后，物料由除病毒主单元1(除病毒主单元1)经过深层过滤流入缓冲体积3；
当除病毒主单元1重量低于下限，结束物料向缓冲体积3的转移；清洗除病毒主单元1；
假如缓冲体积3重量达到设定的重量上限，由DCS控制暂停物料向除病毒主单元1的转移，暂停至缓冲体积3重量重新处于启动区间内之后，物料继续向除病毒主单元1转移。
4.缓冲体积3重量及液位以及
Figure PCTCN2021083516-appb-000003
Process控制阳离子交换层析主单元：
当缓冲体积3重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积3流入阳离子交换层析主单元。DCS系统发送指令启动阳离子交换 层析主单元。在一实施方案中，在使物料由缓冲体积3流入阳离子交换层析主单元之前，DCS系统根据电导传感器数据判断缓冲体积3电导率值是否在设定目标范围内，并根据需要进行电导调节操作，使电导率值达到设定范围的±0.1-0.5mS/cm，稳定1-5min。
当阳离子交换层析主单元进入上样步骤后，DCS系统给在线目标蛋白定量检测系统发送启动蛋白量累计指令，开始实时计算上样蛋白量，并将计算结果发送给DCS系统。
当上样量达到蛋白累计设定值时，DCS系统给
Figure PCTCN2021083516-appb-000004
Process发送指令，停止上样，进入后续层析步骤。
假如缓冲体积3重量低于下限后，在线目标蛋白定量检测系统仍未达到蛋白累计设定值，则判断缓冲体积3为空，由DCS系统给
Figure PCTCN2021083516-appb-000005
Process发送指令，停止上样，进入后续层析步骤。
阳离子交换层析过程中，物料由阳离子交换层析主单元流入缓冲体积4。
假如缓冲体积4重量达到设定的重量上限，由DCS控制
Figure PCTCN2021083516-appb-000006
Process暂停阳离子交换层析运行，暂停至缓冲体积4重量重新处于启动区间内之后，
Figure PCTCN2021083516-appb-000007
Process继续运行。
5.缓冲体积4、缓冲体积5重量及液位以及
Figure PCTCN2021083516-appb-000008
Process控制阴离子交换层析主单元：
当缓冲体积4重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积4流入阴离子交换层析主单元。在一实施方案中，在使物料由缓冲体积4流入阴离子交换层析主单元之前，DCS程序根据在线pH和电导传感器数据进行判断，并根据需要调节pH和电导。
当阴离子交换层析主单元进入上样步骤后，DCS系统给在线目标蛋白定量检测系统发送启动蛋白量累计指令，开始实时计算上样蛋白量，并将计算结果发送给DCS系统。
当上样量达到蛋白累计设定值时，DCS系统给
Figure PCTCN2021083516-appb-000009
Process发送指令，停止上样，继续层析步骤。假如缓冲体积4重量低于下限后，在线目标蛋白定量检测系统仍未达到蛋白累计设定值，则判断缓冲体积4为空，由DCS系统给
Figure PCTCN2021083516-appb-000010
Process发送指令，停止上样，继续层析步骤。
阴离子交换层析过程中，物料由阴离子交换层析主单元流入缓冲体积5。
假如缓冲体积5重量达到设定的重量上限，由DCS控制
Figure PCTCN2021083516-appb-000011
Process暂停阴离子交换层析运行，暂停至缓冲体积5重量重新处于启动区间内之后，
Figure PCTCN2021083516-appb-000012
Process继续运行。
6.缓冲体积5、缓冲体积6重量及液位以及除病毒主单元2恒压控制：
当缓冲体积5重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积5流入除病毒主单元2，以进行除病毒过滤。
通过调节泵流速，控制除病毒过滤压力为恒压。
当缓冲体积5重量低于下限，关闭缓冲体积5罐底阀；清洗膜包。
除病毒过滤过程中，物料由除病毒主单元2流入缓冲体积6。
假如缓冲体积6重量达到设定的重量上限，由DCS控制暂停物料向除病毒主单元2的转移，暂停至缓冲体积6重量重新处于启动区间内之后，继续物料向除病毒主单元2的转移。
7.缓冲体积6、缓冲体积7重量及液位以及浓缩和/或换液主单元1恒压控制：
当缓冲体积6重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积6流入浓缩和/或换液主单元1。启动泵开始浓缩，浓缩过程中通过调节泵流速以控制压力恒定。
当缓冲体积6重量低于下限，停止泵，关闭罐底阀，清洗膜包。
假如缓冲体积7重量达到设定的重量上限，由DCS控制泵暂停运行，暂停至缓冲体积7重量重新处于启动区间内之后，泵继续运行。
浓缩过程中，物料由浓缩和/或换液主单元1流入缓冲体积7。
8.缓冲体积7、缓冲体积8重量及液位以及浓缩和/或换液主单元2恒压控制：
当缓冲体积7重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积7流入浓缩和/或换液主单元2。启动泵开始在线切向流换液。控制切向流换液单元进口处的压力为恒压。
当缓冲体积7重量低于下限，关闭罐底阀，清洗膜包。
物料由浓缩和/或换液主单元2流入缓冲体积8，并作为产品输出。
在一优选的实施方案中：
由缓冲体积1流入亲和层析主单元的料流包含一个或多个亚流；
由亲和层析主单元流入缓冲体积2的料流包含多个亚流；
由缓冲体积2流入除病毒主单元1的料流包含一个亚流；
由除病毒主单元1流入缓冲体积3的料流包含多个亚流；
由缓冲体积3流入阳离子交换层析主单元的料流包含一个亚流；
由阳离子交换层析主单元流入缓冲体积4的料流包含一个或多个亚流；
由缓冲体积4流入阴离子交换层析主单元的料流包含一个亚流；
由阴离子交换层析主单元流入缓冲体积5的料流包含一个亚流；
由缓冲体积5流入除病毒主单元2的料流包含一个亚流；
由除病毒主单元2流入缓冲体积6的料流包含一个亚流；
由缓冲体积6流入浓缩和/或换液主单元1的料流包含一个亚流；
由浓缩和/或换液主单元1流入缓冲体积7的料流包含一个亚流；
由缓冲体积7流入浓缩和/或换液主单元的料流包含一个亚流2；
由浓缩和/或换液主单元2流入缓冲体积8的料流包含一个亚流。
有益效果
在生物制药行业的生产应用中，连续操作仍以“仓对仓”方式，作为单个、断开的CM单元操作实施，并通过这样的方式实现工艺设计的较高维度上的连续制造。仓对仓方式仍然需要持续在CM单元操作之间对物料进行管理和转移，具有批次生产的部分特征，因此仅能部分地实现连续制造的空-时经济性(space-time economy)。
本发明提供了一种整合的、自动化的、全连续化的单抗纯化系统，其特征在于(1)采用了自动控制系统与所有设备进行通信连接，实现了对于过程的调节和控制。(2)实现了亲和层析、低pH病毒灭活、阳离子交换层析、阴离子交换层析、除病毒过滤、超滤浓缩、超滤换液整个纯化过程的全连续化生产。(3)采用PAT对生产过程中的关键质量属性进行在位或在线检测，并通过自动控制系统实现反馈控制。
本发明以简单、成本较低、易于实施和维护的在线目标蛋白定量检测系统实现了PAT。其中，在位UV吸光度检测设备采用1个流通池实现了0.1-10mm的光程变化，结构更简单，可变光程范围相对较大，可测量浓度范围更广。
本发明的在线目标蛋白定量检测系统实时计算单位时间流经目标蛋白总量。其任选地与在线HPLC装置配置于同一层析主单元，能够及时提供上样量、产品纯度相关的信息。实现了在目标蛋白纯化的过程中，对目标蛋白进行在线定量检测以及根据检测结果控制目标蛋白的上样载量，并且在需要时通过反馈控制及时调整纯化参数以确保产品质量。
本发明通过特定主单元上游的缓冲罐的重量来控制该主单元的启动和停止，实现了在不需要确切知道上游下游罐料液总量的条件下，自动进行单元操作。
亲和层析主单元上游设置缓冲罐，可以在实现重量控制的同时，设置重量保护，避免料液耗尽时泵吸入空气导致层析柱损坏。重量控制的方法比现有技术常用的采用气泡感应控制上样终点的方法更加的稳定和可靠，采用气泡感应控制上样终点的方法常常会因为澄清的细胞收获液中的细小气泡(由于流体在管路中碰撞，管路存在空气，或管路存在死角等产生)而导致上样提前结束，存在一定的风险。
当在亲和层析阶段采用多柱位连续层析方法，可以对复杂的原料进行快速判断和处理，保证该CM单元操作能够连续输出具有可靠质量属性的产品，而在其后的层析步骤中，实现了相对简单的工艺。对可选地使用在线HPLC检测电荷异构体含量，对产品质量实现了更完善的控制。整个工艺控制过程相对简单，稳定性高，适用于广泛的工艺条件。
通过本发明的连续纯化系统，靶分子的纯化时间可大幅缩短，纯化效率大幅提高，同时，由于连续、封闭和自动化运行，大大降低了产品污染风险，提高了生物药物纯化设备所占的空间的利用率，减小了设备所需空间，大大降低生产成本和提高产能。
实施例
以下将通过具体实施例来进一步描述本发明的技术方案。应注意，所述实施例仅为示例性，而非对本发明保护范围的限制。本发明还可有其他实施方案，或能够以多种方式实践或进行。除非另有说明，本文中所有的百分比、份数、比值等均是按重量计。
仪器、试剂和材料
Figure PCTCN2021083516-appb-000013
PCC系统来自GE Healthcare。亲和层析填料(MabSelect Sure LX)来自GE。阳离子交换层析填料(型号：Capto S)来自GE。阴离子交换层析填料(型号：Capto Q)来自GE。SPTFF膜包来自Pall。ILDF膜包来自Pall。在线HPLC设备来自Agilent，型号1260，配备UV检测器。
本发明的其他试剂均为可商购的，例如可以购自默克化工。
实施例1抗体纯化系统
本发明的纯化系统可通过不同方式实施，一个示例性方式如下连续纯化单抗的纯化系统，其可以通过图2所示方式实现：
本实施例的纯化系统包括图2所示缓冲体积1至浓缩和/或换液主单元2，包括图2中的ii至xv示出的物料流动过程。
图2中的过程可以描述为如下步骤1-8，其中操作物料流入/流出缓冲体积的执行器为传输泵和各个Tank底部的罐底阀。
每个缓冲体积配置有重量传感器和液位传感器。除病毒罐(除病毒主单元1)配置有pH传感器、电导传感器、重量传感器和液位传感器。每个缓冲体积以及除病毒罐分别独立地设定一个料液下限和一个料液上限(用重量传感器检测值衡量或重量传感器和液位传感器检测值共同衡量)。以仅有重量传感器进行检测为例，当重量传感器的检测值达到或低于所述重量下限时认为该缓冲体积不适宜再为下游主单元提供料液，从而停止料液向该主单元转移并在适当时停止下游主单元的运行；当重量传感器的值达到或高于所述重量上限时认为该缓冲体积不再适宜再接受上游主单元输入的料液，从而停止上游主单元的运行。
对于每个缓冲体积分别独立地设定一个料液检测区间，用于判断是否可以启动紧邻的下游主单元，该重量区间称为启动该缓冲体积的紧邻的下游主单元的区间，简称为该缓冲体积的启动区间。启动区间的下限即该缓冲体积的料液下限，启动区间的上限小于或等于该缓冲体积的料液上限。
1.作为原料的CCCF暂存于缓冲体积1(Tank0)中，即图中i。
2.Tank0以及缓冲体积2(Tank1)重量及液位控制亲和层析主单元：
当Tank0重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积1通过一个或多个亚流流入亲和层析主单元，即图中ii。
由DCS系统启动PCC程序，运行亲和层析方法。
在亲和层析过程中，物料由亲和层析主单元通过多个亚流流入缓冲体积2(Tank1)，即图中iii。
当Tank0重量低于下限，由DCS系统向PCC发送Digital I/O信号，PCC收到信号后，结束上样，进入结束步骤。PCC程序结束后，亲和层析单元操作完成。
假如Tank1重量达到设定的重量上限，由DCS控制PCC暂停运行，暂停至Tank1重量重新处于启动区间内之后，PCC继续运行。
3.Tank1、Tank2以及Tank3重量及液位控制除病毒主单元1：
当Tank1重量达到设定的重量下限，启动搅拌。
当Tank1重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积2通过一个亚流流入除病毒主单元1(Tank2)，即图中iv。
当Tank2重量达到设定的重量下限，启动搅拌。Tank1料液转移至Tank2传输完成后，稳定1-2分钟，根据pH传感器数据，DCS系统判断Tank2的pH值是否在需要的范围内，然后根据需要由自动化控制的泵操作加酸或者加碱，进行低pH病毒灭活操作。
当Tank1重量低于下限，停止搅拌，结束物料向除病毒主单元1的转移。
Tank2的低pH病毒灭活操作完成后，停止搅拌，物料由除病毒主单元1(Tank2)经过深层过滤通过一个或多个亚流流入缓冲体积3(Tank3)，即图中v。
当Tank2重量低于下限，结束物料向Tank3的转移，启动清洗泵，清洗Tank2。
假如Tank3重量达到设定的重量上限，由DCS控制暂停物料向除病毒主单元1的转移，暂停至Tank3重量重新处于启动区间内之后，物料继续向除病毒主单元1转移。
4.Tank3重量及液位以及
Figure PCTCN2021083516-appb-000014
Process控制阳离子交换层析主单元：
当Tank3重量达到设定的重量下限，启动搅拌。
当Tank3重量及液位到达设定值时，根据电导传感器数据，DCS系统判断Tank3电导率值是否在设定目标范围内，并根据需要进行电导调节操作，使电导率值达到设定范围的±0.1-0.5mS/cm，稳定1-5min。停止搅拌，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积3通过一个亚流流入阳离子交换层析主单元，即图中vi。DCS系统发送指令启动阳离子交换层析主单元。
当阳离子交换层析主单元进入上样步骤后，DCS系统发送启动蛋白量累计指令，蛋白质浓度计算设备开始实时计算上样蛋白量，并将计算结果发送给DCS系统。
当上样量达到蛋白累计设定值时，DCS系统给
Figure PCTCN2021083516-appb-000015
Process发送指令，停止上样，进入后续层析步骤(例如洗脱)。
假如Tank3重量低于下限后，在线目标蛋白定量检测系统仍未达到蛋白累计设定值，则判断Tank3为空，由DCS系统给
Figure PCTCN2021083516-appb-000016
Process发送指令，停止上样，进入后续层析步骤。
阳离子交换层析过程中，物料由阳离子交换层析主单元通过一个或多个亚流流入缓冲体积4(Tank4)，即图中vii。
假如Tank4重量达到设定的重量上限，由DCS控制
Figure PCTCN2021083516-appb-000017
Process暂停阳离子交换层析运行，暂停至Tank4重量重新处于启动区间内之后，
Figure PCTCN2021083516-appb-000018
Process继续运行。
5.Tank4、Tank5重量及液位以及
Figure PCTCN2021083516-appb-000019
Process控制阴离子交换层析主单元：
当Tank4重量达到设定的重量下限，启动搅拌。
当Tank4重量及液位到达设定值时，DCS程序根据在线pH和电导传感器数据进行判断，并根据需要调节pH和电导。管理系统发出指令，指示执行器进行操作，使物料由缓冲体积4通过一个亚流流入阴离子交换层析主单元，即图中viii。
当阴离子交换层析主单元进入上样步骤后，DCS系统发送启动蛋白量累计指令，蛋白质浓度计算设备开始实时计算上样蛋白量，并将计算结果发送给DCS系统。
当上样量达到蛋白累计设定值时，DCS系统给
Figure PCTCN2021083516-appb-000020
Process发送指令，停止上样，继续层析步骤。假如Tank4重量低于下限后，在线目标蛋白定量检测系统仍未达到蛋白累计设定值，则判断Tank4为空，由DCS系统给
Figure PCTCN2021083516-appb-000021
Process发送指令，停止上样，继续层析步骤。
阴离子交换层析过程中，物料由阴离子交换层析主单元通过一个亚流流入缓冲体积5(Tank5)，即图中ix。
假如Tank5重量达到设定的重量上限，由DCS控制
Figure PCTCN2021083516-appb-000022
Process暂停阴离子交换层析运行，暂停至Tank5重量重新处于启动区间内之后，
Figure PCTCN2021083516-appb-000023
Process继续运行。
6.Tank5、Tank6重量及液位以及除病毒主单元2恒压控制：
当Tank5重量达到设定的重量下限，启动搅拌。
当Tank5重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积5通过一个亚流流入除病毒主单元2，以进行除病毒过滤，即图中x。
通过调节泵流速，控制除病毒过滤压力为恒压。
当Tank5重量低于下限，关闭Tank5罐底阀；清洗膜包。
除病毒过滤过程中，物料由除病毒主单元2通过一个亚流流入缓冲体积6(Tank6)，即图中xi。
假如Tank6重量达到设定的重量上限，由DCS控制暂停物料向除病毒主单元2的转移，暂停至Tank6重量重新处于启动区间内之后，继续物料向除病毒主单元2的转移。
7.Tank6、Tank7重量及液位以及浓缩和/或换液主单元1恒压控制：
当Tank6重量达到设定的重量下限，启动搅拌。
当Tank6重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积6通过一个亚流流入浓缩和/或换液主单元1，即图中xii。启动泵开始浓缩，浓缩过程中通过调节泵流速以控制压力恒定。
当Tank6重量低于下限，停止搅拌，停止泵，关闭罐底阀，清洗膜包。
假如Tank7重量达到设定的重量上限，由DCS控制泵暂停运行，暂停至Tank7重量重新处于启动区间内之后，泵继续运行。
浓缩过程中，物料由浓缩和/或换液主单元1通过一个亚流流入缓冲体积7(Tank7)，即图中xiii。
8.Tank7、Tank8重量及液位以及浓缩和/或换液主单元2恒压控制：
当Tank7重量达到设定的重量下限，启动搅拌。
当Tank7重量及液位到达设定值时，管理系统发出指令，指示执行器进行操作，使物料由缓冲体积7通过一个亚流流入浓缩和/或换液主单元2，即图中xiv。启动泵开始在线切向流换液。控制切向流换液单元进口处的压力为恒压。
当Tank7重量低于下限，停止搅拌，关闭罐底阀，清洗膜包。
物料由浓缩和/或换液主单元2通过一个亚流流入缓冲体积8(Tank8)，并作为产品输出，即图中xv。
管理系统
纯化系统的管理系统包括工控机和DCS系统。上述连续纯化单抗的纯化系统由分布式控制系统(DCS)整体控制，其采用多种通信协议与设备通信，包括Profibus-DP，OPC，Modbus-RTU，4-20mA信号等，控制程序运行在DCS系统的主控制器或副控制器中。设备产生的数据(包括泵、搅拌器、pH传感器、压力传感器、UV传感器、电导传感器、重量传感器、液位传感器等)将被DCS系统采集，并进行存储。
PAT技术
1.在位UV吸光度检测器用于上样载量控制
DCS系统根据服务技术元件(执行器与传感器)提供的数据，确定步骤运行状态、层析介质的载样状态和预先设置的算法，向执行器(例如泵或阀)发出指令，推动料流在流路中流动或停止。在现有批次生产工艺中，需要将低pH病毒灭活收集液送检单抗浓度，然后根据低pH病毒灭活收集液重量，阳离子交换层析柱体积来计算上样量，需耗时4-5h。采用在位控制后，这部分时间可全部节省。
在位UV吸光度检测器包括：流通池，紫外线照射装置和光谱仪。流通池包括溶液流通道接口和光通道接口。在位UV吸光度检测器在纯化系统中的配置方式参见图3至图5。
图3至图5示出：
流通池10，包括横向的溶液流通道接口(11,12)和纵向的光通道接口(13,14)，所述溶液流通道接口(11,12)与层析装置20，所述光通道接口(13,14)与所述蛋白浓度检测计算装置30连接；该流通池10连接在线目标蛋白定量检测系统100多个设备，含目标蛋白的溶液从系统100的前端设备经过该流通池到达系统100的后端设备；
层析装置20，将含目标蛋白的溶液接入所述在线目标蛋白定量检测系统100，并对流经的含目标蛋白的溶液进行蛋白分离，获得含目标蛋白的溶液；层析装置20将所述含目标蛋白的溶液中的其他成分去除，所述含目标蛋白的溶液中的其他成分包括一些其他蛋白的杂质；
蛋白浓度检测计算装置30，对所述含目标蛋白的溶液的浓度进行检测，并实时计算所述含目标蛋白的溶液的蛋白总量；
蛋白质载量控制设备40，与所述蛋白浓度检测计算装置30和所述层析装置20通信连接，根据所述蛋白总量控制所述层析装置20的上样载量。计算所述含目标蛋白的溶液的蛋白总量后，蛋白质载量控制设备40将根据接收到的该信息控制所述层析装置20的上样载量。具体的说，在所述含目标蛋白的溶液的蛋白总量达到预设值后，停止目标蛋白的溶液上样，由此实现了在线目标蛋白定量检测系统100对目标蛋白的在线定量检测。在一优选的实施方案中，所述目标蛋白为单克隆抗体(以下简称“单抗”)。即在线目标蛋白定量检测系统100实现了在线单抗定量检测。
所述层析装置20包括：
层析设备21，与所述流通池10的溶液流通道接口(11,12)连接，将所述含目标蛋白的溶液接入所述在线目标蛋白定量检测系统100；
至少一个层析柱22，与所述流通池19的溶液流通道接口连接(11,12)，对流经的所述含目标蛋白的溶液进行蛋白分离，将所述含目标蛋白的溶液中的其他成分去除，获得目标蛋白溶液。
层析柱22可以包括多个，设置在不同的位置，当用于检测进入层析柱22前的蛋白浓度，并控制上样量时，流通池10设置于层析柱22在流通管道上游的位置，当需要用于检测层析柱22流出溶液中蛋白浓度时，流通池10可以放在层析柱22下游的位置。如在单抗纯化过程中，将流通池10接入层析装置20管路(例如GE
Figure PCTCN2021083516-appb-000024
设备)中，并位于层析柱22前，层析柱22是对蛋白进行分离的一种柱型装置，含目标蛋白的溶液流过该柱，使蛋白与其他成分分离。由此，蛋白质载量控制设备40将根据接收到的该信息控制所述层析装置20的上样载量，具体是在单抗层析过程中对层析柱22载量进行控制。
参见图4，本发明的蛋白浓度检测计算装置30包括：紫外线照射装置31，与所述流通池10的光通道接口(上端接口)13连接，照射流经所述流通池10的所述含目标蛋白的溶液；所述紫外线照射装置31的发光光源包括氙灯、氘灯、LED灯或者汞灯；所述紫外线照射装置31的发光光源的发射光波长范围为200nm-400nm；
光谱仪32，与所述流通池10的光通道接口(下端接口)14连接，吸收所述紫外线照射装置31照射流经所述流通池10的所述含目标蛋白的溶液的紫外线，并将所述光谱仪32的紫外吸收值数据传送到蛋白质浓度计算设备33；紫外线照射装置31照射所述含目标蛋白的溶液，所述含目标蛋白的溶液吸收紫外线，光谱仪32检测所述含目标蛋白的溶液吸收紫外线的强度，并将该紫外吸收值数据传送到蛋白质浓度计算设备33；所述光谱仪32包括：紫外光谱仪32，紫外-可见光谱仪32；所述光谱仪32的吸光值范围为在0-2AU。优选的，所述紫外线照射装置31的发射光波长范围为270nm-320nm；所述光谱仪32的吸光值范围为在0.05AU-1.6AU；
蛋白质浓度计算设备33，接收所述光谱仪32的紫外吸收值数据，并根据预设的标定的标准曲线，实时计算所述蛋白质浓度以及单位时间流经流通池10的目标蛋白总量；并将所述目标蛋白总量发送到所述蛋白质载量控制设备40；优选的，蛋白质浓度计算设备33为工控机。
其中所述流通池10、蛋白浓度检测计算装置30中的紫外线照射装置31和光谱仪32及其之间的连接即为本发明的在位UV吸光度检测器。
本发明的在位UV吸光度检测器检测时间在10微秒-10秒。其配置于汇聚层入口处(例如阳离子交换层析主单元/阴离子交换层析主单元)。
在层析装置20运行时，蛋白质浓度计算设备33会根据实时测量的浓度，层析设备21上样流速，及上样时间，实时计算累计的上样量，并将该数据即时传给DCS系统。DCS系统将该数据与事先设定的层析柱22上样载量进行对比，如果两者相等，则DCS系统会控制层析设备21停止上样，进入后续的纯化步骤。从而防止上样量超过层析柱22载量。
在一实施方案中，实际操作时首先在采用缓冲液清洗层析装置20时，对紫外光谱仪32进行调零，以便进行浓度测量。从层析装置20流出的含目标蛋白的溶液经过流通池10时，产生紫外吸收峰，其吸收强度与含目标蛋白的溶液浓度的关系在一定范围内符合朗伯比尔定律，可由公式1计算得到。
Figure PCTCN2021083516-appb-000025
公式1中c是单抗浓度,A是紫外吸光值，￡为消光系数，L是流通池10光程。对于已知的含目标蛋白的溶液，如单抗样品，其摩尔消光系数，流通池10光程均为定值。因此可根据紫外吸收值A计算单抗浓度c。光谱仪32的紫外吸收值数据传输至工控机，由工控机根据事先标定的标准曲线，实时计算出单抗浓度。单位时间內，流过流通池10的单抗总量，可以由公式2进行计算：
Figure PCTCN2021083516-appb-000026
公式2中，V是含目标蛋白的溶液流速，m是单抗总量，t为时间。含目标蛋白的溶液流速由层析设备21实时流速得到，时间积分运算由工控机进行实时计算。实时计算出的单抗总量，由工控机发送给DCS系统，用于对层析设备21上样载量进行控制。工控机发送给DCS(Distributed Control System,分布式计算机控制系统)。系统之间的通信协议采用工业通信协议(ModBusRTU)。层析装置20和蛋白浓度检测计算装置30之间采用OPC通信协议。
2.HPLC在线检测电荷异构体含量
HPLC在线检测主要用于阳离子纯化步骤中。现有工艺采用离线检测电荷异构体，耗时20-24h，且需要等待检测结果才能决定纯化步骤的合样区间，降低了生产效率。而采用HPLC在线检测之后，大大缩短或消除了等待时间，提升了效率。采用在位检测的方法，相比批次生产工艺可节约19-23h。
在线HPLC装置的具体配置参见实施例4的“在线HPLC装置”部分。
实施例2亲和层析主单元
在本实施例中，层析装置选用GE
Figure PCTCN2021083516-appb-000027
多柱纯化系统(
Figure PCTCN2021083516-appb-000028
PCC)，其中包括三个层析柱。每根层析柱前后各有一个UV检测器，根据特定层析柱进样料液的UV信号和流出物UV信号判断该层析柱是否达到预设的上样终点。PCC系统仅能检测料液浓度的变化，并以此估算填料载样状态。管理系统根据
Figure PCTCN2021083516-appb-000029
PCC系统包含的程序向执行器(例如泵或阀)发出指令，推动料流在流路中流动或停止。
实施例3除病毒主单元1
除病毒主单元1为低pH病毒灭活系统，其主要包括低pH病毒灭活罐(除病毒主单元1(Tank2))，安装于罐上的pH传感器，及分别连接至酸罐和碱罐的加酸泵和加碱泵。除病毒主单元1(Tank2)与缓冲体积2(Tank1)通过泵相连，并可根据程序设定自动通过酸/碱泵调节亲和层析收集液的pH值。病毒灭活完成后，中和回调至设定pH值，料液经过深层过滤后由除病毒主单元1(Tank2)流出至缓冲体积3(Tank3)。
DCS系统判断缓冲体积3(Tank3)电导率值是否在设定目标范围内，如不在范围内，则进行电导调节。
实施例4阳离子交换层析主单元
1.层析装置
在本实施例中，层析装置可以选用多柱纯化系统。层析装置包括一个层析柱。层析柱的入口配置有本发明的在位UV吸光度检测器。管理系统根据相邻步骤的运行情况、离子交换层析介质的结合状态和预先设置的算法向执行器(例如泵或阀)发出指令，推动料流在流路中流动或停止。
2.在位UV吸光度检测器
在位UV吸光度检测器配置于阳离子交换层析柱的入口处。
参见图5至图7，流通池10的溶液流通道接口(11,12)包括设置于所述流通池横向两端的溶液流入接口11和溶液流出接口12；所述光通道接口(13,14)包括设置于所述流通池纵向两端的上端接口13和下端接口14，并且在所述流通池10的中部具有供所述含目标蛋白的溶液通过的光径口15；优选的，所述的流通池10为可变光程流通池；所述 可变光程流通池的光程由调整所述光径口15的大小来实现，调整可通过游标卡尺进行调整；具体的，所述光径口15由所述流通池10的中部设置的两根光纤151的间距构成，所述两根光纤151的间距为0.1mm至10mm。调整两根光纤151的间距，即可改变所述可变光程流通池10的光程，而改变流通池10光程是为了可以调整仪器可测量单抗浓度的范围，其可测量的单抗浓度范围在100g/L至0.lg/L之间。
参见图6至图7，蛋白浓度检测计算装置30包括：流通池10的溶液流入接口11与层析设备21连接，流通池10的溶液流出接口12与层析柱22连接；所述上端接口13与所述紫外线照射装置31连接，所述下端口14与所述光谱仪32连接。由层析设备21提供样品流(即所述含目标蛋白的溶液流)。样品经过流通池10后，从溶液流出接口12流出，溶液流出接口12与层析柱22连接。上端接口13为光纤接口，通过光纤151与光源连接。下端接口14为光纤接口，通过光纤151与光谱仪32连接。紫外线照射装置31的光源发射的光，被流通池10中流经两根光纤151之间的单抗吸收，光谱仪32检测透过的光。单抗样品浓度与紫外吸收强度成正比，因此可计算出单抗浓度。而且当光纤在流路中，由于流过光纤狭缝中的料液很少，大部分料液从光线周围空间通过，不会造成流动阻力。此外，所述两根光纤151通过螺纹接口与所述流通池10连接，并且其与上端接口13连接的所述光纤151与所述紫外线照射装置31的发光光源连接；发光光源优选采用HeraeusFiberLight(氘灯)，氘灯发光更稳定。与溶液流出接口12连接的所述光纤151与所述光谱仪32连接。此外，所述光纤151为抗紫外石英光纤，所述光纤151的芯径为450微米；所述两根光纤151通过SMA905接头与所述流通池10连接。
该流通池10材质为不锈钢，两根光纤151采用螺纹接口(例如SMA905)与流通池10连接。其中上端接口13连接的一根光纤151连接光源，用于提供特定波长的紫外光(例如280nm)，另一根与下端接口14连接的光纤151连接光谱仪32,用于将单抗吸收后的光信号传输至光谱仪32,从而得到吸光值A。光谱仪32选用OCEAN-FX-UV-VIS-ES单波长光谱仪，可手动任意设定波长(200nm-850nm)；光谱仪32的检测时间在10微秒-10秒，光谱仪32的检测范围在0-2AU,流通池10光程可由改变两根光纤151之间的间距来进行调节，通常可在0.1毫米至10毫米之间调节。其可测量的单抗浓度在100g/L至0.1g/L之间。
可选地，发光光源在光源机中，光源机为紫外线照射装置31，紫外线照射装置31照射流通池10的两光纤151之间的蛋白溶液。可以选择的发光光源包括但不限于氙灯，氘灯，LED灯，汞灯等，其发射光波长范围应包含200nm-400nm,优选波长在270nm-320nm之间。以及可以选择的光谱仪32包括但不限于紫外光谱仪，紫外-可见光谱仪等，其吸光值范围通常在0-2AU之间。为防止光谱仪32出现信号饱和导致灵敏度下降，通常其可用的吸光值范围为0.05-1.6AU。普通石英光纤在较强紫外光(300nm以下)的照射下会在光纤内形成缺陷中心，导致光纤透射率下降。光纤为抗紫外石英光纤，芯径450微米，材质为抗紫外辐照石英XSR190-1100nm,所述两根光纤通过接头SMA905 与流通池10连接。
在线目标蛋白定量检测系统100进行目标蛋白定量检测，具体的：
(1)检测例1
流通池10：不锈钢流通池，可变光程(游标卡尺手动调整至约0.5mm，即两根光纤151之间的距离)；样品：Mab1
1)系统平衡后调零，建立标准曲线；选取了4个点建立标准曲线
表1
Figure PCTCN2021083516-appb-000030
2)标准曲线准确度测定
表2
Figure PCTCN2021083516-appb-000031
3)在线目标蛋白定量检测系统100对单位时间内的蛋白累计量测定
表3
Figure PCTCN2021083516-appb-000032
测定结果参见表1～表3，由此可见在线目标蛋白定量检测系统100的3个循环次数上样过程UV值基本稳定，蛋白累计误差较小。本次测试中实现了UV调零和蛋白累计调零的自动控制。
(2)检测例2
流通池10：不锈钢流通池，可变光程(游标卡尺手动调整至约0.15mm，即两根光纤151之间的距离)；样品：Mab2
1)系统平衡后调零，建立标准曲线；选取了4个点建立标准曲线
表4
Figure PCTCN2021083516-appb-000033
2)标准曲线准确度测定
表5
Figure PCTCN2021083516-appb-000034
3)在线目标蛋白定量检测系统100对单位时间内的蛋白累计量测定
表6
Figure PCTCN2021083516-appb-000035
测定结果参见表4～表6，由此可见2个Cycle上样过程UV值基本稳定，蛋白累计误差较小，本次测试中实现了UV调零和蛋白累计调零的自动控制。
由此可见，在线目标蛋白定量检测系统100对单位时间内的蛋白累计量测定、测量速度快，最快可达到1ms。其他方案在1s或以上。可测量范围广，单个流通池10就可测量0.1g/L至100g/L浓度范围。可以与DCS系统进行数据通信；可以对一定时间内流过流通池10的单抗量进行计算。
3.在线HPLC装置
如图8所示，在线HPLC装置中，进样环与六通阀2号、5号位置连接，HPLC泵与六通阀1号位置连接，HPLC分析柱与六通阀6号位置连接，六通阀4号位置通过取样管路与纯化系统的流路连接。在线HPLC检测的方法为：
(1)取样(Bypass/Loop loading)：DCS发送指令使六通阀切换，样品由4号位置流入 六通阀，经5号位置流入进样环。在一定流速下，经过预设的载样时间后，进样环的载样过程完成，DCS发送指令使六通阀切换，进入(2)样品分析步骤。
(2)样品分析(Mainpass/Column loading)：HPLC泵推动步骤(1)中装载于进样环的样品由5号位置流入六通阀，经6号位置流入HPLC分析柱。
(3)数据处理与样品收集：
检测器收集不同时间点的检测信号，传输至Matlab软件，并计算目标分子的纯度，判断该样品是否符合合样标准。计算得到的样品纯度数据如果小于产品纯度设定值，则将相应流分(fraction)转化为废液。如果样品纯度大于等于产品纯度设定值，则将相应流分收集至产品收集罐(即，本步骤层析主单元的下游缓冲体积，例如实施例1所示的缓冲体积4(Tank4))。由于实现了基于对产品纯度的实时测量来合并流分，产品质量的可变性得以最小化。
4.层析过程
(1)当阳离子交换层析进入平衡步骤时，在线目标蛋白定量检测系统进入待机状态。当平衡步骤进行约20min后，确保UV流通池已冲洗干净，对在线目标蛋白定量检测系统进行调零。
(2)当阳离子程序进入上样步骤后，DCS系统给在线目标蛋白定量检测系统发送启动蛋白量累计指令，开始实时计算上样蛋白量，并将计算结果发送给DCS系统。
(3)当上样量达到蛋白累计设定值时，DCS系统发送指令，停止上样；或者
如上一步骤输出的料液已全部用于上样，而在线目标蛋白定量检测系统检测结果仍未达到蛋白累计设定值，则由DCS系统发送指令，停止上样，并开始进行下一步骤。
(4)以结合-洗脱模式进行阳离子交换层析。洗脱过程中，对洗脱的样品进行HPLC在位检测。在洗脱步骤开始时，由DCS系统对HPLC发送启动信号，开始进行HPLC分析。
(5)HPLC方法为自主开发的快速CEX-HPLC方法，检测时间仅为4.7min。阳离子洗脱保留时间为6min，因此每个洗脱体积(column volume,CV)均可以检测到。
(6)洗脱收集液采用在线HPLC进行实时分析。
(7)HPLC的分析结果传输至Matlab软件，当样品检测完成后，由自主开发的程序按照给定的合样标准进行合样计算(例如酸性异构体峰<30％，主峰>60％，碱性异构体峰<15％)。所述程序与DCS系统之间通过所述程序包含的数据接口相连接。本领域技术人员可以根据实际情况，用可商购的工作站、程序包或应用来代替本步骤采用的自主开发的程序。
(8)根据程序给出的合样计算结果(例如合样区间为P2-P11，或者例如P2至P11分别进行合样操作)在缓冲体积4(Tank4)进行样品合样。
实施例5阴离子交换层析主单元
在本实施例中，层析装置可以选用多柱纯化系统。层析装置包括一个层析柱。层析柱的入口配置有本发明的在位UV吸光度检测器。管理系统根据相邻步骤的运行情况、离子交换层析介质的结合状态和预先设置的算法向执行器(例如泵或阀)发出指令，推动料流在流路中流动或停止。
实施例6除病毒主单元2
除病毒主单元2为除病毒过滤系统(纳滤)，包括恒压泵及除病毒过滤器，恒压泵的一个进口与缓冲体积5(Tank5)连接，通过调节隔膜泵流速控制恒定在设定压力(29psi)下进行除病毒过滤，过滤后样品流出除病毒主单元2。
实施例7浓缩和/或换液主单元
1.浓缩和/或换液主单元1(单向切向流过滤(SPTFF))
浓缩和/或换液主单元1为超滤浓缩系统，由恒压泵及单向切向流浓缩膜包构成，恒压泵的一个进口与缓冲体积6(Tank6)相连，在设定压力(1.5Bar)下进行浓缩，浓缩后样品流出浓缩和/或换液主单元1。
浓缩结束后，以预定的冲洗体积冲洗膜包。
2.浓缩和/或换液主单元2(切向流换液)
浓缩和/或换液主单元2为超滤换液系统，由泵及单向在位洗滤膜包构成，泵进口与缓冲体积7(Tank7)相连，在程序设定条件下进行单向洗滤。
以蠕动泵为调节料流流动或停止的执行器。控制切向流换液单元进口处的压力为恒压(15-25psi)。进料蠕动泵和置换液蠕动泵的转速比按照恒定比例进行调节(约4-5倍)。
换液结束后，以预定的冲洗体积冲洗膜包。
换液后样品即为最终产品溶液。
实施例8存在汇聚层的纯化系统
当关注汇聚层的汇聚作用时，由于层析主单元是提高物料纯度的关键步骤，因此汇聚层的汇聚作用主要通过各个层析主单元来体现。在实施例1所述的纯化系统的基础上，本实施例设置包含汇聚层的纯化系统。
8.1层析主单元中的亚单元设置
亲和层析主单元包括7个亚单元，其通过三个层析柱分别以结合-洗脱模式运行2或3个周期来实现。当亲和层析主单元包括7个亚单元时，其输出流包含7个亚流。
阳离子交换层析主单元包括3个亚单元，其通过一个层析柱以结合-洗脱模式运行3个周期来实现；或者包括1个亚单元，其通过一个层析柱以结合-洗脱模式运行1个周期来实现。
阴离子交换层析主单元包括1个亚单元，其通过一个层析柱以流穿模式运行1个周期来实现。
8.2存在一个汇聚层的纯化系统
存在一个汇聚层(阳离子交换层析主单元作为汇聚层)的纯化系统示意图参见图9。其中：
阳离子交换层析主单元包括1个亚单元；
亲和层析主单元输出的7个亚流沿纯化系统的管路流动至阳离子交换层析主单元出口时，已汇聚为1个亚流。即，阳离子交换层析主单元输出流仅包含1个亚流。
在该系统中，每个亲和层析从单元通常各自独立地分别由样品检测系统进行检测，因此亲和层析主单元这一单元操作需要样品检测系统运行7次。阳离子交换层析主单元仅配置一套检测器，相应地，该单元一个运行周期仅需要样品检测系统运行1次。
8.3存在二个汇聚层的纯化系统
存在二个汇聚层(阳离子交换层析主单元、阴离子交换层析主单元作为汇聚层)的纯化系统示意图参见图10。其中：
阳离子交换层析主单元包括3个亚单元；
亲和层析主单元输出的7个亚流沿纯化系统的管路流动至阳离子交换层析主单元出口时，已汇聚为3个亚流。其中阳离子交换层析主单元的3个亚流沿纯化系统的管路流动至阴离子交换层析主单元出口时，已汇聚为1个亚流。

Claims (22)

  1. 一种用于进行连续纯化的纯化系统，其包含
    (1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；
    (2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个病毒灭活亚单元；和
    (3)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元；
    在每个主单元的上游或下游各自独立地任选存在的一个或更多个缓冲体积；每个所述主单元及缓冲体积之间通过可使料液流通的管路连接，
    其中，每个缓冲体积配置至少一个重量传感器和/或至少一个液位传感器，所述重量传感器和液位传感器与管理系统通信连接；
    所述管理系统根据所述重量传感器和/或液位传感器的信号对所述纯化系统进行启动、继续和停止的控制；
    所述纯化系统纯化的靶分子是蛋白；
    所述浓缩和/或换液单元是用于实施以连续操作进行的超滤或透析的单元，在所述浓缩和/或换液单元中，包含样品的流股的流动方向与滤出方向不同。
  2. 权利要求1的纯化系统，其中，
    亲和层析主单元上游存在至少一个缓冲体积，并且任一主单元上游和下游均存在至少一个缓冲体积；所述管理系统根据重量及液位信号来控制进行以下操作：
    I.当亲和层析主单元上游的缓冲体积的重量传感器反馈值和/或液位传感器反馈值达到预设值时，纯化系统运行启动；
    II.当任一主单元上游的缓冲体积的重量传感器反馈值和/或液位传感器反馈值达到预设值时，启动该主单元运行；
    III.当任一主单元上游的缓冲体积的重量传感器反馈值和/或液位传感器反馈值为设定下限值时，停止料液向该主单元转移，当单元操作完成后该主单元停止运行；
    IV.当任一主单元下游的缓冲体积的重量传感器和液位传感器反馈值均达到设定上限值时，该主单元停止运行。
  3. 权利要求1或2的纯化系统，其依次包含：
    (1)至少一个亲和层析主单元，其中每个亲和层析主单元各自独立地包含一个或更多个亲和层析亚单元；
    (2)至少一个病毒灭活主单元，其中病毒灭活主单元各自独立地包含一个或更多个 病毒灭活亚单元；
    (3)至少一个阳离子交换层析主单元，所述阳离子交换层析主单元各自独立地包含一个或更多个阳离子交换层析亚单元；
    (4)至少一个阴离子交换层析主单元，所述阴离子交换层析主单元各自独立地包含一个或更多个阴离子交换层析亚单元；和
    (5)至少一个浓缩和/或换液主单元，其中每个浓缩和/或换液主单元各自独立地包含一个或更多个浓缩和/或换液亚单元。
  4. 权利要求1-3中任一项的纯化系统，其中
    在至少一个上游主单元的至少一个亚单元目标料液流出所述主单元之后，所述管理系统允许至少一个所述上游主单元的紧邻下游主单元的至少一个亚单元启动运行；并且
    所述管理系统允许任一所述上游主单元的第一亚单元目标料液流出所述上游主单元之后，所述上游主单元与其紧邻下游主单元运行时间至少一部分重叠。
  5. 权利要求1-4中任一项的纯化系统，其中
    在所述纯化系统流通管道的相对于至少一个层析单元的上游或下游的位置，配置有一个或多个在位UV吸光度检测器，其与管理系统通信连接；
    管理系统根据所述在位UV吸光度检测器在位检测的信号来控制所述层析单元的上样终点。
  6. 权利要求1-5中任一项的纯化系统，其包括操作层和自动化层，其中，操作层包括所有工艺技术元件(PTU)和服务技术元件(STU)，自动化层包括用于数据采集和处理的元件；并且
    操作层包含按以下顺序连接的三个层：(1)原料输入层；(2)汇聚层；(3)产品输出层；
    其中汇聚层具备以下I、II、III：
    I.向汇聚层输入的物料已经经过至少一个纯化步骤，在用于表征物料属性的参数组成的空间中，不同时间点向汇聚层输入的物料的属性分散的程度相对于原料而言已经降低；并且根据预先设定的参数要求，汇聚层将物料进行进一步汇聚，使得由汇聚层输出的物料在预先设定的一个或多个维度上具备比向汇聚层输入的物料更高的汇聚性；
    II.以N表示某一层析主单元中包括的层析亚单元数目，则所述使层析亚单元数目减少的趋势可以用下式(1)和/或(2)表示：
    (1)N(汇聚层)≤N(汇聚层上游相邻层析主单元)；
    (2)N(汇聚层下游相邻纯化主单元)≤N(汇聚层上游相邻层析主单元)；
    其中所述汇聚层下游相邻纯化主单元是层析主单元、除病毒主单元或浓缩和/ 或换液主单元；
    III.汇聚层中的层析主单元包含一个层析亚单元；或者
    汇聚层中的层析主单元包含多个层析亚单元；多个层析亚单元的操作时间相互连续。
  7. 权利要求1-6中任一项的纯化系统，其中
    所述操作层包含一个汇聚层，其为阳离子交换层析主单元。
  8. 权利要求1-7中任一项的纯化系统，其中
    所述操作层包含二个汇聚层，分别为阳离子交换层析主单元和阴离子交换层析主单元。
  9. 权利要求8的纯化系统，其中阳离子交换层析主单元以结合-洗脱模式进行层析；阴离子交换层析主单元以流过模式进行层析。
  10. 权利要求6-9中任一项的纯化系统，其中
    在所述汇聚层的入口处，配置有一个或多个在位UV吸光度检测器，其与管理系统通信连接；
    管理系统根据所述在位UV吸光度检测器在位检测的信号来控制汇聚层层析单元中的层析介质的上样终点。
  11. 权利要求1-10中任一项的纯化系统，其中
    所述浓缩和/或换液步骤为切向流过滤，优选单向切向流过滤；或者，所述浓缩和/或换液步骤为单向切向流过滤和/或在位透析。
  12. 权利要求1-11中任一项的纯化系统，其中
    所述除病毒主单元选自除病毒过滤、病毒灭活或其组合；
    其中，病毒灭活的方式优选为低pH灭活。
  13. 权利要求1-12中任一项的纯化系统，其中
    所述亲和层析主单元为多柱纯化系统；
    所述多柱位层析系统包含至少两个层析柱，每个层析柱各自独立地处于以下状态之一：a.加载步骤、b.非加载步骤和c.静息状态；
    在所述纯化系统流通管道的相对于至少一个层析柱的上游或下游的位置，配置有一个或多个在位UV吸光度检测器，其与管理系统通信连接；
    所述管理系统根据层析柱的结合状态和预先设置的算法发出指令，控制层析柱在所述状态a、b和c之间切换。
  14. 权利要求1-13中任一项的纯化系统，其依次包括缓冲体积1、亲和层析主单元、缓冲体积2、除病毒主单元1、缓冲体积3、阳离子交换层析主单元、缓冲体积4、阴离子交换层析主单元、缓冲体积5、除病毒主单元2、缓冲体积6、浓缩和/或换液主单元1、缓冲体积7、浓缩和/或换液主单元2，纯化过程中物料的流动如以下操作iii至xv：
    i.作为原料的澄清的细胞收获液暂存于缓冲体积1中；
    ii.物料由缓冲体积1通过一个或多个亚流流入亲和层析主单元；
    iii.物料由亲和层析主单元通过多个亚流流入缓冲体积2；
    iv.物料由缓冲体积2通过一个亚流流入除病毒主单元1；
    v.物料由除病毒主单元1通过多个亚流流入缓冲体积3；
    vi.物料由缓冲体积3通过一个亚流流入阳离子交换层析主单元；
    vii.物料由阳离子交换层析主单元通过一个或多个亚流流入缓冲体积4；
    viii.物料由缓冲体积4通过一个亚流流入阴离子交换层析主单元；
    ix.物料由阴离子交换层析主单元通过一个亚流流入缓冲体积5；
    x.物料由缓冲体积5通过一个亚流流入除病毒主单元2；
    xi.物料由除病毒主单元2通过一个亚流流入缓冲体积6；
    xii.物料由缓冲体积6通过一个亚流流入浓缩和/或换液主单元1；
    xiii.物料由浓缩和/或换液主单元1通过一个亚流流入缓冲体积7；
    xiv.物料由缓冲体积7通过一个亚流流入浓缩和/或换液主单元2；
    xv.物料由浓缩和/或换液主单元2通过一个亚流流入缓冲体积8，并作为产品输出；
    优选地，缓冲体积1、缓冲体积2、缓冲体积3、缓冲体积4、缓冲体积5、缓冲体积6、缓冲体积7分别为缓冲罐；管理系统根据重量及液位信号来控制进行如权利要求2所述的操作。
  15. 权利要求1-14中任一项的纯化系统，其中
    所述在位UV吸光度检测器包含：
    流通池，包括溶液流通道接口和光通道接口，所述溶液流通道接口与层析装置连接，所述光通道接口与所述蛋白浓度检测计算装置连接；
    紫外线照射装置，与所述流通池的光通道接口连接，照射流经所述流通池的所述含目标蛋白的溶液；
    光谱仪，与所述流通池的光通道接口连接，检测所述紫外线照射装置照射流经所述流通池的所述含目标蛋白的溶液的紫外线强度，并将紫外吸收值数据传送到蛋白质浓度计算设备。
  16. 权利要求15的纯化系统，其中
    所述流通池的溶液流通道接口包括设置于所述流通池横向两端的溶液流入接口和溶液流出接口；所述光通道接口包括设置于所述流通池纵向两端的上端接口和下端接口，并且在所述流通池的中部具有供所述含目标蛋白的溶液通过的光径口；
    所述流通池的溶液流入接口与所述层析设备连接，所述流通池的溶液流出接口与所述层析柱连接；所述上端接口与所述紫外线照射装置连接，所述下端接口与所述光谱仪连接。
  17. 权利要求15或16的纯化系统，其中
    所述流通池为可变光程流通池；所述可变光程流通池的光程由调整所述光径口的大小调整；
    所述光径口由所述流通池的中部设置的两根光纤的间距构成，所述两根光纤的间距为0.1mm至10mm。
  18. 权利要求16或17的纯化系统，其中
    所述流通池上端接口和下端接口为光纤接口，所述两根光纤通过所述上端接口和下端接口与所述流通池连接，并且与所述上端接口连接的所述光纤与所述紫外线照射装置的发光光源连接；与所述下端接口连接的所述光纤与所述光谱仪连接。
  19. 权利要求18的纯化系统，其中
    所述光纤为抗紫外石英光纤，所述光纤的芯径为450微米；所述两根光纤通过SMA905接头与所述流通池连接。
  20. 权利要求15-19中任一项的纯化系统，其中
    所述紫外线照射装置的发光光源包括氙灯、氘灯、LED灯或者汞灯；所述紫外线照射装置的发光光源的发射光波长范围为200nm-400nm；
    所述光谱仪包括：紫外光谱仪，紫外-可见光谱仪；所述光谱仪的吸光值范围为0-2AU。
  21. 权利要求20所述的纯化系统，其中
    所述紫外线照射装置的发射光波长范围为270nm-320nm；
    所述光谱仪的吸光值范围为在0.05AU-1.6AU；
    所述目标蛋白为单抗。
  22. 一种纯化靶分子的方法，所述方法通过权利要求1-21中任一项的系统完成。
PCT/CN2021/083516 2020-03-30 2021-03-29 连续纯化生物制药产品的系统和方法 WO2021197248A1 (zh)

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