WO2021197249A1 - 一种用于靶分子连续纯化的系统和方法 - Google Patents

一种用于靶分子连续纯化的系统和方法 Download PDF

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WO2021197249A1
WO2021197249A1 PCT/CN2021/083517 CN2021083517W WO2021197249A1 WO 2021197249 A1 WO2021197249 A1 WO 2021197249A1 CN 2021083517 W CN2021083517 W CN 2021083517W WO 2021197249 A1 WO2021197249 A1 WO 2021197249A1
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main unit
liquid
purification
concentration
chromatography
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PCT/CN2021/083517
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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 CN202011404668.4A external-priority patent/CN114573657A/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 WO2021197249A1 publication Critical patent/WO2021197249A1/zh

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    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes

Definitions

  • the invention relates to the field of biopharmaceuticals, and in particular to a system for continuous purification of biopharmaceutical products.
  • the target molecules of biopharmaceuticals are usually protein macromolecules, and the production process is relatively complicated, including multiple purification steps and strict cleaning, 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, and continuous production technology is difficult to apply to the commercial production of biopharmaceutical products. In this case, the production efficiency is not high, the production capacity is insufficient, and the product price is expensive.
  • 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 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 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 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 signals of the weight sensor and the liquid level sensor;
  • one of the concentration and/or liquid exchange main units is an ultrafiltration concentration system, which is composed of a constant pressure pump and a unidirectional tangential flow concentration membrane package;
  • the target molecule purified by the purification system is a protein, preferably an antibody or fusion protein containing an Fc fragment.
  • the present invention also provides a purification system for continuous purification as described above, wherein the control of the purification system is jointly controlled by a weight sensor and a liquid level sensor,
  • the present invention also provides a purification system for continuous purification as described above, in which at least one affinity chromatography subunit of at least one affinity chromatography main unit flows out of said After the affinity chromatography main unit, the management system allows at least one virus inactivation subunit of the at least one virus inactivation main unit to start operation; at least one virus inactivation subunit of the at least one virus inactivation main unit After the unit target feed liquid flows out of the virus inactivation main unit, the management system allows at least one concentration and/or liquid exchange subunit of at least one of the concentration and/or liquid exchange main units to start operation, and the management system After allowing the first subunit target liquid of the affinity chromatography main unit to flow out of the affinity chromatography main unit, the virus inactivation main unit and the affinity chromatography main unit at least partly overlap the running time; After the management system allows the target liquid of the first subunit of the virus inactivation main unit to flow out of the virus inactivation main unit, the running time of the virus inactivation main unit and
  • 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 subunit 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 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.
  • 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).
  • 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.
  • 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 (such as a buffer tank, a storage 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., on 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.
  • concentration and/or liquid exchange step refers to the step of reducing the volume of liquid in the fluid material and/or exchanging the buffer system after the "polishing" step of antibody purification. Generally, such a step facilitates the preservation of the purified antibody.
  • concentration and/or fluid exchange main unit may, for example, comprise an ultrafiltration or dialysis unit.
  • 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 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 the following purification system for continuous purification of monoclonal antibodies, which can be implemented in the manner shown in Figure 1:
  • the purification system of this embodiment includes the affinity chromatography main unit shown in FIG. 1 to the concentration and/or liquid exchange main unit 2, including operations iii to xiv in FIG. 1.
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from buffer volume 1 into the affinity chromatography main unit through one or more substreams .
  • the material flows into the buffer volume 2 (Tank1) from the affinity chromatography main unit through multiple substreams.
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 2 (Tank1) through a subflow into the virus removal main unit 1 ( Tank2).
  • the material flows into the buffer volume 3 (Tank3) from the virus removal main unit 1 through one or more subflows.
  • 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 through a sub-flow.
  • the material flows into the buffer volume 4 (Tank4) from the main unit of cation exchange chromatography through one or more substreams.
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from the buffer volume 4 into the main unit of anion exchange chromatography through a subflow.
  • the material flows into the buffer volume 5 (Tank5) from the anion exchange chromatography main unit through a substream.
  • 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 through a sub-flow.
  • the material flows into the buffer volume 6 (Tank6) from the virus removal main unit 2 through a subflow.
  • the management system issues instructions to instruct the actuator to operate, so that the material flows from the buffer volume 6 through a sub-flow into the concentration and/or liquid exchange main unit 1 .
  • the material flows into the buffer volume 7 (Tank7) from the main unit 1 for concentration and/or liquid exchange through a subflow.
  • the management system issues an instruction to instruct the actuator to operate, so that the material flows from the buffer volume 7 through a sub-flow into the main unit 2 for concentration and/or liquid exchange. .
  • 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.
  • 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.
  • Embodiment 2 Affinity Chromatography Main Unit
  • GE is selected as the chromatography device Multi-column purification system ( PCC), which includes three chromatography columns.
  • the main unit of affinity chromatography includes 9 sub-units, which are realized by running three chromatography columns in a bind-elution mode for 3 cycles.
  • 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.
  • Embodiment 3 Virus removal main unit 1
  • 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 the pH value of the affinity chromatography collection solution can be automatically adjusted by the acid-base pump according to the program setting.
  • 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 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 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 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 injection loop is connected to the six-port valve position No. 2 and No. 5
  • the HPLC pump is connected to the six-port valve position No. 1
  • the HPLC analytical column is connected to the six-port valve position No. 6, and the six-way valve position passes through
  • the sampling pipeline is connected with the flow path of the purification system.
  • 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 sampling 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 DCS system After the cation program enters the sample loading step, when the sample load reaches the set value (such as sample loading time or volume setting), the DCS system sends an instruction to stop the sample loading; or
  • the DCS system will send an instruction to stop sample loading and start the next step.
  • HPLC analysis results are transferred to the Matlab software.
  • the self-developed program will perform the sample combination calculation according to the given sample standard (for example, acid isomer peak ⁇ 30%, main peak>60%, Alkaline 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 anion exchange chromatography main unit includes one or more subunits, which are realized by running one or more chromatography columns in a flow-through mode for one or more cycles.
  • 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, including a constant pressure pump and a virus removal filter. One inlet of the constant pressure pump is connected to buffer volume 5 (Tank5). The virus removal filtration is performed under a set pressure, and the sample flows out after filtration. In addition to the virus main unit 2.
  • Embodiment 7 Concentration and/or liquid exchange main unit
  • 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 carried out 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.

Abstract

生物制药领域中一种连续纯化生物制药产品的系统。通过传感器的监控控制纯化环节的启动和停止，同时，通过切向流浓缩技术使得整个纯化过程处在封闭、连续、自动化的状态下运行，并且无需中间环节停止管路运行，取样监测质量，大大提高的生产效率，缩短纯化时间。

Description

一种用于靶分子连续纯化的系统和方法
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202011404668.4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蛋白质大分子，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，连续化生产技术难以应用到生物制药产品的商业化生产中，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。
在一些方面，本发明提供一种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；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；
并且，其中一个浓缩和/或换液主单元为超滤浓缩系统，由恒压泵及单向切向流浓缩膜包构成；
所述纯化系统纯化的靶分子是蛋白，优选为含Fc片段的抗体或融合蛋白。
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，所述纯化系统的控制由重量传感器和液位传感器共同控制，
(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；
(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；
(3)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。
在一些实施方案中，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。
有益效果
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。
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“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 PCTCN2021083517-appb-000001
系统。Warikoo等人报道了
Figure PCTCN2021083517-appb-000002
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。
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“浓缩和/或换液步骤”是指抗体纯化“抛光”步骤之后，减少流体物料中的液体体积和/或交换缓冲体系的步骤。通常，这样的步骤利于纯化后的抗体的保存。浓缩和/或换液主单元可以例如包含超滤或透析的单元。
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。
附图说明
图1.靶分子连续纯化系统的料流流动过程
图2.在线HPLC装置
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 PCTCN2021083517-appb-000003
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 单抗纯化系统
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1所示方式实现：
本实施例的纯化系统包括图1所示亲和层析主单元至浓缩和/或换液主单元2，包括图1中的操作iii至xiv。
图1中：
i.作为原料的CCCF暂存于缓冲体积1(Tank0)中；
ii.当缓冲体积1(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。
iii.物料由亲和层析主单元通过多个亚流流入缓冲体积2(Tank1)。
iv.当缓冲体积2(Tank1)重量和液位到达设定值时，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(Tank1)通过一个亚流流入除病毒主单元1(Tank2)。
v.物料由除病毒主单元1通过一个或多个亚流流入缓冲体积3(Tank3)。
vi.当缓冲体积3(Tank3)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(Tank4)。
viii.当缓冲体积4(Tank4)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(Tank5)。
x.当缓冲体积5(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。
xi.物料由除病毒主单元2通过一个亚流流入缓冲体积6(Tank6)。
xii.当缓冲体积6(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。
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(Tank7)。
xiv.当缓冲体积7(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。
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(Tank8)，并作为产品输出。
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。
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 PCTCN2021083517-appb-000004
多柱纯化系统(
Figure PCTCN2021083517-appb-000005
PCC)，其中包括三个层析柱。亲和层析主单元包括9个亚单元，其通过三个层析柱以结合-洗脱模式运行3个周期来实现。管理系统根据
Figure PCTCN2021083517-appb-000006
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。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。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.在线HPLC装置
如图2所示，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。
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。
3.层析过程
(1)当阳离子交换层析完成平衡步骤时，启动蛋白上样步骤。
(2)当阳离子程序进入上样步骤后，当上样量达到设定值(如上样时间或体积设定)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，则由DCS系统发送指令，停止上样，并开始进行下一步骤。
(3)以结合-洗脱模式进行阳离子交换层析。
(4)洗脱收集液采用在线HPLC进行实时分析。
(5)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(例如酸异构体峰<30％，主峰>60％，碱异构体峰<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。
(6)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。阴离子交换层析主单元包括1个或更多个亚单元，其通过一个层析柱或更多个层析柱以流穿模式运行1个或更多个周期来实现。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，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，在设定压力下进行除病毒过滤，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。

Claims (8)

  1. 一种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；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；
    并且，其中一个浓缩和/或换液主单元为超滤浓缩系统，其包括泵及切向流浓缩膜包，优选单向切向流浓缩膜包；
    所述纯化系统纯化的靶分子是蛋白，优选为含Fc片段的抗体或融合蛋白。
  2. 根据权利要求1所述的用于进行连续纯化的纯化系统，所述纯化系统的控制由重量传感器和/或液位传感器共同控制，
    (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；
    (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；
    (3)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. 根据权利要求1或2所述的用于进行连续纯化的纯化系统，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的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/或换液主单元的运行时间至少一部分重叠。
  4. 根据权利要求1至3任一项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。
  5. 根据权利要求1至4任一项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。
  6. 根据权利要求1至5任一项所述的用于进行连续纯化的纯化系统，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。
  7. 根据权利要求1至5任一项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。
  8. 一种用于靶分子的连续纯化方法，其使包含靶分子的澄清原液流入如权利要求1至7任一项所述的至少一个亲和层析主单元上游紧邻的缓冲体积，并使所述澄清原液经权利要求1至7任一项所述的纯化系统分离纯化的步骤。
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