CN114573657A

CN114573657A - System and method for continuous purification of target molecules

Info

Publication number: CN114573657A
Application number: CN202011404668.4A
Authority: CN
Inventors: 巩威; 陈然; 姚彬
Original assignee: Shanghai Henlius Biotech Inc
Current assignee: Shanghai Henlius Biotech Inc
Priority date: 2020-12-02
Filing date: 2020-12-02
Publication date: 2022-06-03

Abstract

The present invention relates to the field of biopharmaceuticals, and in particular to a system for continuous purification of biopharmaceutical products by target molecules. The start and stop of purification link is controlled through the control of sensor, simultaneously, makes whole purification process be in the operation under sealed, continuous, the automatic state through the tangential flow concentration technique to need not the intermediate link and stop the pipeline operation, sample monitoring quality, the production efficiency who improves greatly shortens the purification time.

Description

System and method for continuous purification of target molecules

Technical Field

The present invention relates to the field of biopharmaceuticals, and in particular to a system for continuous purification of biopharmaceutical products.

Background

Biopharmaceutical target molecules are usually protein macromolecules, and the production process is complex and comprises multiple purification steps and strict procedures of cleaning, sterilization and virus removal. However, due to the complexity of the production of biological products, and in order to meet the technical requirements and regulatory specifications of pharmaceutical product production, multiple indexes need to be detected and controlled in the production process, and the continuous production technology is difficult to be applied to the commercial production of the biological pharmaceutical products, which results in low production efficiency, insufficient productivity and high product price.

WO2020148119a1 discloses a method for converting a batch purification process of a monoclonal antibody into a continuous purification process. Wherein the antibody downstream process adopts protein A (protein A) affinity chromatography, cation exchange chromatography, anion exchange chromatography, virus filtration and ultrafiltration/dialysis. Protein a affinity chromatography using Bio SMB chromatography with 5 columns, a continuous feed stream and a continuous eluent stream can be generated. CEX employs a parallel batch mode (batch mode), and both viral filtration and ultrafiltration/dialysis employ a batch mode (batch mode).

CN106794424A discloses a control system and method for use with a connection system of a chromatography process unit or the like in fluid communication with a tangential flow filtration process unit.

Disclosure of Invention

The present invention provides a purification system for continuous purification for continuous, closed and automated purification of target molecules.

In some aspects, the present invention provides a purification system for performing continuous purification, comprising, in order:

(1) at least one affinity chromatography master unit, wherein each affinity chromatography master unit independently comprises one or more affinity chromatography subunits;

(2) at least one virus-inactivation master unit, wherein the virus-inactivation master units each independently comprise one or more virus-inactivation subunits; and

(3) at least one concentration and/or exchange liquid main unit, wherein each concentration and/or exchange liquid main unit independently comprises one or more concentration and/or exchange liquid sub-units;

one or more buffer volumes, each independently optionally present, upstream or downstream of each main unit; each of said main units and buffer volumes are connected by a line for the passage of feed liquid,

wherein each buffer volume is provided with at least one weight sensor and at least one liquid level sensor, which are in communication connection with a management system;

the management system controls the starting, continuing and stopping of the purification system according to signals of the weight sensor and the liquid level sensor;

one of the concentration and/or liquid change main units is an ultrafiltration concentration system and consists of a constant pressure pump and a unidirectional tangential flow concentration membrane;

the target molecule purified by the purification system is a protein, preferably an antibody or fusion protein containing an Fc fragment.

In other aspects, the invention also provides a purification system for continuous purification according to the preceding, the control of the purification system being controlled by both a weight sensor and a level sensor,

(1) when the feedback value of the weight sensor and/or the feedback value of the liquid level sensor of the buffer volume at the upstream of the affinity chromatography main unit reach a preset value, the purification system is started;

(2) when the feedback value of the weight sensor and/or the feedback value of the liquid level sensor of the buffer volume at the upstream of any main unit is a set lower limit value, stopping the transfer of the feed liquid to the main unit, and stopping the operation of the main unit after the unit operation is finished;

(3) when the feedback values of the weight sensor and the liquid level sensor of the buffer volume downstream of any main unit reach the set upper limit values, the main unit stops running.

In some embodiments, there is at least one buffer volume upstream of the affinity chromatography main units, and at least one buffer volume upstream and downstream of any main unit.

In other aspects, the present invention further provides a purification system for continuous purification according to the above, wherein the management system allows at least one virus-inactivation subunit of at least one of the virus-inactivation main units to start operation after the target feed liquid of at least one of the affinity chromatography subunits of at least one of the affinity chromatography main units flows out of the affinity chromatography main unit; after the target feed liquid of at least one virus inactivation subunit of at least one virus inactivation main unit flows out of the virus inactivation main unit, the management system allows at least one concentration and/or liquid change subunit of at least one concentration and/or liquid change main unit to start running, and after the target feed liquid of the first subunit of the affinity chromatography main unit flows out of the affinity chromatography main unit, the virus inactivation main unit and the affinity chromatography main unit run time at least partially overlap; the management system allows the operation time of the virus inactivation main unit and the concentration and/or liquid exchange main unit to at least partially overlap after the first subunit target liquid of the virus inactivation main unit flows out of the virus inactivation main unit.

It should be understood that when it is described that the running times of more than one unit overlap at least partially, it indicates that the overlapping occurs on the time axis between the running times of the units, and it is not limited that the running of more than one unit starts at the same time point or ends at the same time point. In an embodiment, the expression "at least a portion of the operating times overlap" 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 of the operating times of a unit.

In other aspects, the present invention also provides a purification system for performing continuous purification according to the foregoing, further comprising at least one cation exchange chromatography main unit and/or at least one anion exchange chromatography main unit between the affinity chromatography main unit and the concentration and/or liquid exchange main unit, the cation exchange chromatography main units each independently comprising one or more cation exchange chromatography sub-units, and the anion exchange chromatography main units each independently comprising one or more anion exchange chromatography sub-units.

In other aspects, the invention also provides a purification system for carrying out a continuous purification according to the preceding description, comprising in sequence:

(2) at least one virus-inactivation master unit, wherein the virus-inactivation master units each independently comprise one or more virus-inactivation subunits;

(3) at least one cation exchange chromatography main unit, each independently comprising one or more cation exchange chromatography subunits;

(4) at least one anion exchange chromatography main unit, each independently comprising one or more anion exchange chromatography subunits; and

(5) at least one concentration and/or exchange liquid main unit, wherein each concentration and/or exchange liquid main unit independently comprises one or more concentration and/or exchange liquid sub-units.

In other aspects, the present invention further provides a purification system for continuous purification according to the above, wherein the management system allows at least one subunit of at least one upstream main unit, which is immediately downstream of the upstream main unit, to start operation after at least one subunit target feed liquid of the upstream main unit flows out of the main unit; and said management system allows said upstream master cell to overlap at least a portion of its immediate downstream master cell operating time after said upstream master cell has had its first subunit target feed solution flowing out of said upstream master cell.

In other aspects, the invention also provides a purification system according to the preceding for performing a continuous purification, which is a continuous, closed, automated separation and purification of a target molecule.

In addition, the invention also provides a continuous purification method for target molecules, which is completed by the system of the invention. In one embodiment, the method further comprises the steps of flowing a clarified cell harvest (CCCF) comprising a target molecule into a buffer volume immediately upstream of the at least one affinity chromatography main unit as described above, and isolating and purifying the CCCF via a purification system as described above.

Advantageous effects

By the continuous purification system, the purification time of target molecules can be greatly shortened, the purification efficiency is greatly improved, meanwhile, due to continuous, closed and automatic operation, the product pollution risk is greatly reduced, the utilization rate of the space occupied by the biological medicine purification equipment is improved, the space required by the equipment is reduced, the production cost is greatly reduced, and the productivity is improved.

Detailed Description

The invention will be described in further detail below with the understanding that the terminology is intended to be in the nature of words of description rather than of limitation.

General terms and definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application will control. When an amount, concentration, or other value or parameter is expressed in terms of a range, preferred range, or upper preferable numerical value and lower preferable numerical value, it is understood that any range defined by any pair of upper range limits or preferred numerical values in combination with any lower range limits or preferred numerical values is specifically disclosed, regardless of whether the range is specifically disclosed. Unless otherwise indicated, numerical ranges set forth herein are intended to include the endpoints of the ranges and all integers and fractions (decimal) within the range.

Unless the context indicates otherwise, singular references such as "a", "an", include plural references. The expression "one or more" or "at least one" may denote 1, 2, 3, 4, 5, 6, 7, 8, 9 or more(s).

The terms "about" and "approximately," when used in conjunction with a numerical variable, generally mean that the value of the variable and all values of the variable are within experimental error (e.g., within 95% confidence interval for the mean) or within ± 10% of the specified value, or more.

The term "metering ratio" refers to the ratio of various substances according to a certain weight.

The terms "optional" or "optionally present" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The expressions "comprising" or similar expressions "including", "containing" and "having" and the like which are synonymous are open-ended and do not exclude additional, unrecited elements, steps or components. The expression "consisting of …" excludes any element, step or ingredient not specified. The phrase "consisting essentially of …" means that the scope is limited to the specified elements, steps or components, plus optional elements, steps or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" covers the expressions "consisting essentially of …" and "consisting of …".

The terms "process step" or "unit operation" are used interchangeably herein to refer to the use of one or more methods or devices in a purification process to achieve a result. Examples of processing steps or unit operations that may be used in the purification process include, but are not limited to, clarification, chromatography, virus inactivation, and concentration and/or exchange. It should be understood that each process step or unit operation may employ more than one step or method or apparatus to achieve the desired result for that process step or unit operation.

The term "material conversion" is broadly defined as the conversion of a particular material under the influence of physical, chemical or biological conditions to another material having different properties. This conversion may be manifested as a change in physical properties (e.g., melting, dissolution, etc.), a change in chemical properties (e.g., a chemical reaction, such a reaction may be conducted under biological conditions, such as an enzyme-catalyzed reaction), or a change in composition of the material as a mixture (e.g., a purification process). Wherein, the material refers to the object of production and processing and the product of production and processing. By "material" is meant, in general terms, a material object that is the effect of a production process, covering a time span from the beginning of the production process (raw material) to the end of the production process (final product).

The term "continuous production" or "continuous purification" (CM, or continuous purification) refers to production or purification characterized by a continuous process (also referred to as "continuous process") or continuous operation (continuous operation). Wherein "continuously" means that the starting materials are continuously fed into the reactor and the reaction products are also continuously discharged from the reactor. At each time point there is simultaneous material movement as feedstock input and as product output. "continuous production" or "continuous manufacturing" or "continuous purification" allows for continuous, closed and automated separation and purification of target molecules, including at least two CM unit operations running at least partially overlapping in time.

Continuous manufacturing may improve pharmaceutical manufacturing in, for example, the following respects. For example, with integrated processes, steps are reduced, processing time is reduced; the required equipment occupies a small area; support for enhanced development methods (e.g., design quality (QbD) and use of process analysis techniques, use of mathematical models); monitoring the product quality in real time; and provides flexible operation so that process scale-up, scale-down and scale-out can be conveniently performed to accommodate changing supply requirements. Thus, the use of continuous manufacturing for drug production is expected to reduce drug quality issues, reduce manufacturing costs, and increase the chances that patients will receive good quality drugs. Novasep (Pompey, France), Tarpon (Worcester, Mass.), Semba (Madison, Wis.) and GE Healthcare (Piscataway, N.J.) provide several commercial continuous chromatography systems, such as those of GE Healthcare

PCC system. Warikoo et al report

The PCC system is integrated with an upstream continuous bioreactor to form a continuous biological treatment platform for purification of the monoclonal antibody or recombinant enzyme by affinity chromatography.

The "buffer volume" herein has the effect of effecting an adjustment of the distribution of material in the continuous process, providing a buffer to flow material between unit operations at a desired flow rate. The buffer volume may be of any known form, for example a container (such as a buffer tank, a reservoir bag), or an inflatable tube. It will be appreciated that the container may have a fully fixed shape or may be wholly or partially deformable.

The term "surge tank" refers to any container or vessel or bag used between or within processing steps (e.g., when a single processing operation includes more than one step); wherein the output from one step flows through a surge tank to the next step. Thus, a buffer tank is not intended to hold or collect the entire volume of output from a step, as is a collection tank; but instead enables the continuous flow of output from one step to the next.

The term "unit" refers to a device or a part of a device for carrying out a process step or its operation in cycles. The unit is capable of performing a specific function in the purification system. "Unit" includes, but is not limited to, "Master" and "subunit". "master unit" means a set of devices or parts of devices that perform a specific function in a specific step, also understood as a module. Examples of the main unit include, but are not limited to, a chromatography main unit, a concentration and/or exchange liquid main unit, a virus removal main unit, and the like. The main units are connected by flow paths so that the material undergoes the respective process steps during its passage through the respective main units. Preferably, each master unit is operated simultaneously at similar feed rates in order to minimize production time. The "master unit" is made up of one or more "subunits" that perform the same kind of function. "subunit" means a cycle of operation of a device or a part of a device which carries out a process step and can also be expressed as a cycle of operation. For example, any affinity chromatography main unit may comprise one or more affinity chromatography subunits, i.e. one or more affinity chromatography columns each run independently for one or more cycles. Similarly, any of the cation exchange chromatography main unit, the anion exchange chromatography main unit, or the virus removal main unit may comprise one or more subunits. An implementation of a "unit" may be a device known to perform a specific function desired. For example, a chromatography unit may be implemented by any known chromatography device, such as a membrane chromatography device, a chromatography column, beads, magnetic beads, and other stationary phase carriers, a multi-column flow system, a fluidized bed chromatography system, and other chromatography systems, and the like. In a particular embodiment, a chromatography subunit refers to an operational cycle of a chromatography column or a membrane chromatography device, in particular of a chromatography column. As used herein, a unit has one or more elements selected from process technology elements, service technology elements, elements for data acquisition and processing.

Process Technology Units (PTUs) include elements for performing steps, including but not limited to hoses, filters, chromatography columns, vessels, etc., which are not connected to a management system.

The Service Technology Unit (STU) includes all sensors and actuators of the unit. They are connected to the management system through the RIO. The actuators of the STU may be, for example, pumps, valves, and the sensors may be, for example, UV measurements, pressure sensors, weight sensors, or the like.

The elements used for data acquisition and processing are, in the simplest case, remote I/O, or local intelligence, such as Programmable Logic Control (PLC) or PC-based systems with an I/O layer. The basic automation of the unit is performed on a local control. These two system variants are referred to below as RIO (registered input/output).

The term "sub-stream" refers to a stream from a unit that is formed by the output of material to its adjacent downstream units. Wherein a sub-stream is a stream formed by the output of a time-continuous primary material from a sub-unit to its adjacent downstream unit. For example, when the main unit of affinity chromatography is a multi-column continuous affinity chromatography device, one cycle of operation of any one of the columns is an affinity chromatography subunit, and the output stream of each affinity chromatography subunit is used as an input stream of the next step (adjacent downstream units). When multiple affinity chromatography subunits are present, the output stream of an affinity chromatography master unit to an adjacent downstream unit contains multiple sub-streams. This should also be understood for the output streams of other types of main units, such as cation exchange chromatography main units. The output streams of the two subunits are continuous with each other, which means that when the output stream of the first subunit is finished, the output stream of the second subunit is just started, namely, the output streams of the two subunits are continuous in time, and the two output streams can be considered to form a continuous stream together. The case where the output streams of more than two subunits are contiguous to each other should be similarly understood. The operation time of the two subunits being consecutive to each other means that the operation of the second subunit is just started when the operation of the first subunit is finished, i.e. the operation of the two subunits is consecutive in time. The situation where the operating times of two or more subunits are consecutive to each other should be similarly understood. In the case of a flow-through chromatography master unit comprising a plurality of subunits, it will be understood that the output streams of two or more subunits should also be mutually sequential, provided that the operating times of the two or more subunits are sequential therebetween. It will be appreciated that in a typical continuous flow process, the output of the buffer volume to the adjacent downstream unit is fully continuous, which is described herein as an output flow containing only one sub-flow.

One run cycle corresponds to a certain amount of sample input and the progress of the sample output, and the times of sample input and sample output may overlap partially, overlap completely, or not overlap completely. For example, chromatography is performed in a bind-elute mode, wherein one run cycle comprises one (i.e. a certain amount) sample input (bind) -sample output (elute) process, and the time of sample input and sample output in one run cycle of one chromatography column partly overlap or not overlap at all, preferably not overlap at all. In some cases, for example when chromatography is performed in flow-through mode, the time of sample input and the time of sample output, respectively, are completely continuous, in which case a specific length of time may be assigned to a run cycle, said specific length of time corresponding to a specific sample input quantity and/or a specific sample output quantity, and said specific sample input quantity and/or specific sample output quantity corresponding to the quantity of product output in the immediately upstream unit for one run cycle. That is, when dividing the operation cycle of such a device or device portion, the time for the device or device portion to process a sample produced in one operation cycle immediately upstream of the unit and output downstream as a product can be divided into one operation cycle as necessary. Wherein the time for processing the sample generated in one cycle of operation immediately upstream of the unit comprises the time for receiving the sample (loading), such that the time for the start of the cycle of operation is the time for the start of receiving the sample generated in one cycle of operation immediately upstream of the unit. When the time of sample input and the time of sample output, respectively, for a particular master unit are completely continuous, one cycle of operation for that master unit may be defined as the time for processing the sample produced one cycle of operation for the immediately upstream master unit and outputting downstream as product.

In some cases, the end point of one period of time (e.g., one operating cycle) is the same as the start point of another period of time (e.g., another operating cycle), and such a case does not belong to the overlapping of the periods of time, and such a case is also included in the case of the "completely non-overlapping".

It will be appreciated that for continuous production, two or more cycles of operation may be partially overlapping, fully overlapping or fully non-overlapping, preferably partially overlapping or fully non-overlapping, for a particular plant or plant section. For example, in some embodiments, the time for processing the sample produced by one run cycle immediately upstream of the unit in a particular run cycle of the one chromatography column in flow-through mode may partially or completely overlap with the time for outputting the product downstream of another run cycle (e.g., the previous run cycle), and the degree of overlap may be set in advance as needed to achieve continuous production. In other embodiments, there is no overlap at all between a particular run cycle and another run cycle of one chromatography column that performs chromatography in bind-elute mode. In order to achieve continuous production, the sample input times and/or sample output times of the sub-units of a particular master unit can be coordinated as required to achieve a continuation of the master unit sample input time and/or a continuation of the sample output time, or, if required, a continuation of the buffer volume immediately upstream of the master unit sample output time and/or a continuation of the buffer volume immediately downstream of the master unit sample input time can also be achieved by means of the buffer volume.

It will be appreciated that any one device or part of a device may optionally be operated for one or more operating cycles, depending on product requirements, or on sample size, or on the length of time the purification system is operated, and in particular may be controlled by a management system.

In one embodiment, the length of a particular operational cycle of any apparatus or portion of an apparatus (e.g., any component in the apparatus) performing a process technology step can be measured, anticipated, and controlled.

Conventional antibody chromatographic purification steps include: (i) concentration of the target product and removal of harmful impurities (capture), (ii) optionally further removal of large amounts of impurities (enhance), (iii) finally, removal of remaining traces of impurities and undesired structural variants of the target product, such as dimers and multimers of the target product (polish, "polishing"). The discussion herein of the operation of an antibody chromatographic purification unit is primarily directed to affinity chromatography, ion exchange chromatography and does not exclude the use of any known chromatographic means that may be used to purify antibodies, such as mixed mode chromatography, hydrophobic interaction chromatography, size exclusion chromatography, hydroxyapatite chromatography, and any combination of the above. Preferably, the chromatography step is selected from the group consisting of affinity chromatography, ion exchange chromatography and combinations thereof. Purification of the antibody also includes virus removal unit operations. The virus-removing unit operation may be any known method as long as it meets the unit operation product quality requirements 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.

The term "concentration and/or exchange step" refers to a step of reducing the volume of liquid in the fluid material and/or exchanging the buffer system after the antibody purification "polishing" step. Generally, such steps facilitate preservation of the purified antibody. The concentration and/or exchange main unit may for example comprise an ultrafiltration or dialysis unit.

The term "process characterization" refers to the process of characterizing a process. Which includes measuring, categorizing, and evaluating the parameters.

The term "process design space" refers to the design space defined by the ICH Q8 guidelines. Design space refers to the multidimensional combination and interaction of input variables (e.g., material properties) and process parameters that have been proven to provide quality assurance. Thus, the design space is defined by the important and critical process parameters and their acceptable ranges determined from the process characterization studies. This definition cannot be expanded by the process designer at his or her discretion, but requires the industry and regulatory bodies to set forth. The design space of a certain product must be subject to regulatory evaluation and approval. The method of establishing a design space involves process characterization studies and generally involves three key steps: 1. performing a risk analysis to identify parameters for process characterization; 2. design multivariate study protocols using a DoE (design of experiments) experimental design to enable studies to obtain data suitable for understanding and defining the design space; 3. a study plan is performed and the results of the study are analyzed to determine the importance of the parameters and their role in establishing a design space, wherein the impact of the parameters on the CQA is evaluated, process parameters that statistically significantly affect the CQA are evaluated, and process parameters determined to have a significant impact on the CQA are classified as CPP, and acceptable ranges of important and critical process parameters are calculated, e.g., by uncertainty analysis of estimated fault edges. These acceptable ranges collectively define the design space. In general, the design space can be enlarged/reduced by enlarging/reducing the mathematical model.

DoE is generally used to look up a range of instrument operating parameters to see variations in sample preparation and variations in method accuracy.

The term "characterization range" refers to the range examined during process characterization. "Method Operable Design Range (MODR)" refers to a parameter of a process design space that can be changed within a regulatory allowed range.

The term "Quality by Design (QbD)" refers to a systematic development method that performs process Design based on reasonable scientific and Quality risk management, starting from predefined goals, and emphasizes product and process understanding and process control. QbD may facilitate improvements in the manufacturing process within the approved design space (e.g., continuous improvements using PAT tools) without further regulatory scrutiny and may reduce approved process change applications.

The term "Process Analytical Technologies (PAT)" is defined as: a system for designing, analyzing, and controlling production that guarantees end product quality by measuring in real time raw materials, in-process materials (in-process materials), and process Critical Quality Attributes (CQA) and critical performance attributes (FDA PAT guidelines, 2004). The concept of PAT is complementary to the concept of design space. The use of PAT is an integral part of QbD, which provides a means for feedback control of the process based on measurements of CQA.

The desired goal of the PAT framework is to design and develop a well understood process, always ensuring a preset quality at the end of the production process. The process is considered to be well understood when the following is reached: identifying and explaining all key change sources; secondly, the change can be managed through the process; the product quality attribute can be accurately and reliably predicted through a design space established by the used raw materials, process parameters, production, environment and other conditions. The use of PAT allows a more fundamental understanding of the process and therefore a fundamental improvement over traditional biological manufacturing. For example, application of PAT may help to obtain univariate or multivariate statistical process control (SPC or MSPC) models. It should be understood that PAT cannot be implemented by merely improving the analysis techniques, for example, online or in-situ detection of parameters does not mean that PAT is implemented because it does not identify and interpret key sources of variation, nor does it provide assistance in implementing online control to ensure product quality attributes. QbD and PAT are intended only to ensure good product quality and to develop processes that can operate under a wider range of conditions. The selection of CQAs follows the same strategy. However, the use of PAT may cause changes in the means of CPP selection and monitoring, control strategy, relative to the case where PAT is not used, which cannot be anticipated in advance.

The term "Critical Quality Attribute (CQA)" is a physical, chemical, biological property or characteristic that should be within the appropriate limits, ranges or distributions to ensure the desired product quality. In creating design space, it is desirable to have the current production quality control program consistently (efficiently) meet the critical quality attribute range. Implementing the process within the design space allows the product to meet well-defined CQA specifications.

The term "Critical Process Parameter (CPP)" refers to a process parameter in a process that significantly affects CQA.

The term "out of specification (OOS) refers to an out of specification test result, i.e., a test result that does not meet statutory quality standards or enterprise internal control standards. OOS is a resulting deviation and is often caused by production operations. Continuous manufacturing processes generally require consideration of OOS processing strategies in advance for process stability and to ensure product quality. The processing of the OOS generally includes performing a corresponding bias analysis (e.g., whether the error occurred due to an experiment, such as the experiment not meeting system compliance or acceptance criteria for the experiment (or not meeting a portion of the acceptance criteria for the experiment), whether there is an anomaly in the technology/equipment, the material, etc.) and a bias processing flow.

In-line detection is also known as in-situ detection. On-line detection techniques typically require modification of the bioreactor to divert the sample stream. Off-line or off-line detection is a discontinuous analytical method involving sampling or sample pre-treatment. Some documents collectively refer to "in-place" and "online" as online. Accordingly, in some embodiments, the "in-place" detection, "in-place" devices and the "online" detection, "online" devices described herein may each be implemented in an "in-line" or "online" manner. In some particular embodiments, the "in-place" detection or "in-place" devices described herein are implemented in an "in-place" manner.

"upstream" means at a position in the purification system that is forward or upstream of the material flow-through process; "downstream" means at a location in the purification system that is later or downstream in the material flow-through process, throughout the purification system material flow-through process. Whether a particular primary unit or buffer volume is located upstream or downstream is relative, taking the virally inactivated primary unit as an example, in some embodiments, the affinity chromatography primary unit is located upstream of the virally inactivated primary unit, while the concentration and/or exchange primary unit is located downstream of the virally inactivated primary unit.

The 'adjacent unit' of a unit means that no other functional unit exists between the unit and the adjacent unit except for a connecting pipeline, and the adjacent unit can be a main unit or a buffer volume; "immediately adjacent master unit" of a master unit means that no other master unit exists between the master unit and its immediately adjacent master unit except for the connecting lines and the buffer volumes; "immediately upstream master unit" means a master unit immediately upstream of a certain master unit.

"weight sensor" means a sensor that can directly or indirectly sense the weight of a "buffer volume" or "buffer tank"; "level sensor" refers to a sensor that can directly or indirectly sense the level or position of a liquid in a "buffer volume" or "buffer tank".

Drawings

FIG. 1 flow process of a continuous purification system for target molecules

FIG. 2 on-line HPLC apparatus

Examples

The technical solution of the present invention will be further described below by specific examples. It should be noted that the described embodiments are only illustrative and not limiting to the scope of the invention. The invention is capable of other embodiments or of being practiced or carried out in various ways. All percentages, parts, ratios, etc. herein are by weight unless otherwise indicated.

Instruments, reagents and materials

PCC system is from GE Healthcare. Affinity chromatography packing (MabSelect Sure LX) was from GE. The cation exchange chromatography packing (model: Capto S) was from GE. The anion exchange chromatography packing (type: Capto Q) was from GE. SPTFF membranes were from Pall. ILDF membranes were from Pall. The on-line HPLC equipment from Agilent, model 1260 was equipped with a UV detector.

Other reagents according to the invention are commercially available, for example from the merck chemical industry.

EXAMPLE 1 monoclonal antibody purification System

The purification system of the present invention can be implemented in various ways, and one exemplary way is a purification system for continuous purification of monoclonal antibodies, which can be implemented in the manner shown in FIG. 1:

the purification system of this example comprises the affinity chromatography main unit shown in fig. 1 to the concentration and/or exchange liquid main unit 2, including operations iii to xiv in fig. 1.

In fig. 1:

i. CCCF as starting material was buffered in buffer volume 1(Tank 0);

when the weight and level of buffer volume 1(Tank0) reach the set values, the management system issues commands instructing the actuators to operate to flow material from buffer volume 1 through one or more sub-streams into the affinity chromatography main unit.

Material was flowed from the affinity chromatography main unit into buffer volume 2 via multiple sub-streams (Tank 1).

When the weight and level of the buffer volume 2(Tank1) reach the set values, the management system issues commands instructing the actuators to operate so that the material flows from the buffer volume 2(Tank1) through a sub-stream into the virus removal main unit 1(Tank 2).

Material flows from the virus removal main unit 1 into the buffer volume 3(Tank3) via one or more sub-streams.

When the weight and level of buffer volume 3(Tank3) reach the set values, the management system issues commands instructing the actuators to operate to flow material from buffer volume 3 through a sub-stream into the cation exchange chromatography main unit.

Material is flowed from the cation exchange chromatography main unit into buffer volume 4 via one or more sub-streams (Tank 4).

When the weight and level of the buffer volume 4(Tank4) reach the set values, the management system issues commands instructing the actuators to operate so that material flows from the buffer volume 4 through a sub-stream into the anion exchange chromatography main unit.

Material was passed from the anion exchange chromatography main unit through a sub-stream into the buffer volume 5(Tank 5).

x. when the weight and level of the buffer volume 5(Tank5) reach the set values, the management system issues commands instructing the actuators to operate so that the material flows from the buffer volume 5 through a sub-stream into the virus removal main unit 2.

Material flows from the virus removal master unit 2 into the buffer volume 6(Tank6) via a sub-stream.

When the weight and level of the buffer volume 6(Tank6) reach the set values, the management system gives commands instructing the actuators to operate so that the material flows from the buffer volume 6 through a sub-flow into the concentration and/or exchange main unit 1.

Material flows from the concentration and/or exchange main unit 1 via a sub-stream into the buffer volume 7(Tank 7).

When the weight and level of the buffer volume 7(Tank7) reach the set values, the management system gives commands instructing the actuators to operate so that the material flows from the buffer volume 7 through a sub-flow into the concentration and/or exchange main unit 2.

xv. the material is fed from the concentrate and/or change liquid main unit 2 via a sub-stream into the buffer volume 8(Tank8) and is discharged as product.

Management system

The management system of the purification system comprises an industrial personal computer and a DCS. The purification system for continuously purifying the monoclonal antibody is integrally controlled by a Distributed Control System (DCS), various communication protocols are adopted to communicate with equipment, the equipment comprises Profibus-DP, OPC, Modbus-RTU, 4-20mA signals and the like, and a control program runs in a main controller or a secondary controller of the DCS. Data generated by the equipment (including pumps, stirrers, pH sensors, pressure sensors, UV sensors, conductivity sensors, weight sensors, liquid level sensors, etc.) are collected by the DCS system and stored.

Example 2 affinity chromatography Main Unit

In this embodiment, the chromatography device is GE

Multiple column purification System (

PCC) comprising three chromatography columns. The main affinity chromatography unit comprises 9 subunits, which is achieved by running three columns in bind-elute mode for 3 cycles. The management system is based on

The PCC system includes a program that commands an actuator (e.g., a pump or a valve) to move or stop the flow of material in the flow path.

Example 3 Virus removal Master 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 mounted on the Tank, and an acid addition pump and a base addition pump connected to the acid Tank and the base Tank, respectively. The virus-removal main unit 1(Tank2) is connected to the buffer volume 2(Tank1) by a pump, and the pH of the affinity chromatography collection liquid can be automatically adjusted by an acid-base pump according to the program setting. After the virus inactivation is completed, neutralization is adjusted back to a set pH value, and the feed liquid flows out to a buffer volume 3(Tank3) from a virus removal main unit 1(Tank2) after being subjected to deep filtration.

The DCS judges whether the conductivity value of the buffer volume 3(Tank3) is within a set target range, and if not, the conductivity is adjusted.

Example 4 cation exchange chromatography Main Unit

1. Chromatography device

In this embodiment, the chromatography device may be a multi-column purification system. The chromatographic device comprises a chromatographic column. The cation exchange chromatography main unit comprises 3 subunits, which are realized by running a chromatographic column in a binding-elution mode for 3 cycles; or 1 subunit, by running one column for 1 cycle in bind-elute mode. The management system commands an actuator (e.g., a pump or valve) to move or stop the flow of material in the flow path based on the operation of the adjacent step, the binding status of the ion exchange chromatography medium, and a pre-set algorithm.

2. On-line HPLC device

As shown in fig. 2, the on-line HPLC apparatus comprises 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 flow/stop of the sample in the on-line HPLC apparatus is pushed with the pump. The sampling pipeline is a branch on an eluent outlet pipeline of the chromatographic equipment, and a sample enters a sample ring in the six-way valve through the sampling pipeline and then returns to the eluent outlet pipeline. The online HPLC unit sends a signal to and is controlled by the DCS.

In the on-line HPLC device, the sample injection ring is connected with the positions No. 2 and No. 5 of the six-way valve, the HPLC pump is connected with the position No. 1 of the six-way valve, the HPLC analytical column is connected with the position No. 6 of the six-way valve, and the position No. 4 of the six-way valve is connected with the flow path of the purification system through the sampling pipeline. The method for on-line HPLC detection comprises the following steps:

(1) sampling (Bypass/Loop loading): DCS sends an instruction to switch the six-way valve, and the sample flows into the six-way valve from the No. 4 position and flows into the sample injection ring through the No. 5 position. And (3) under a certain flow rate, after the preset sample loading time, completing the sample loading process of the sample loading ring, and sending an instruction by the DCS to switch the six-way valve to enter the step (2) of analyzing the sample.

(2) Sample analysis (Mainpass/Column loading): and (2) pushing the sample loaded on the sample injection ring in the step (1) to flow into the six-way valve from the position 5 and flow into the HPLC analytical column through the position 6 by the HPLC pump.

(3) Data processing and sample collection:

the detector collects detection signals of different time points, transmits the detection signals to Matlab software, calculates the purity of target molecules and judges whether the sample meets the sample combination standard. And if the calculated sample purity data is less than the set value of the product purity, converting the corresponding fraction (fraction) into waste liquid. If the sample purity is equal to or greater than the product purity setpoint, the corresponding fraction is collected in the product collection Tank (i.e., the buffer volume downstream of the chromatography main unit of this step, e.g., buffer volume 4(Tank4) shown in example 1). Variability in product quality is minimized due to the fact that fractions are combined based on real-time measurements of product purity.

3. Chromatography process

(1) When the cation exchange chromatography has completed the equilibration step, the protein loading step is initiated.

(2) After the cation program enters the sample loading step, when the sample loading amount reaches a set value (such as sample loading time or volume setting), the DCS sends an instruction to stop sample loading; or

And if the feed liquid output in the previous step is completely used for sample loading, sending an instruction by the DCS, stopping sample loading, and starting to perform the next step.

(3) Cation exchange chromatography was performed in bind-elute mode.

(4) The eluted pool was analyzed on-line HPLC in real time.

(5) The results of the HPLC analysis were transmitted to Matlab software and when the sample detection was complete, the pooling calculations were performed by an autonomously developed program according to the given pooling standards (e.g. acid isomer peak < 30%, main peak > 60%, base isomer peak < 15%). The program is connected with the DCS system through a data interface contained in the program. One skilled in the art can substitute commercially available workstations, packages, or applications for the self-developed programs employed in this step, as the case may be.

(6) Sample pooling is performed in buffer volume 4(Tank4) according to the results of the pooling calculations given by the program (e.g., pooling intervals of P2-P11, or pooling operations performed separately from P2 to P11, for example).

Example 5 anion exchange chromatography Main Unit

In this embodiment, the chromatography device may be a multi-column purification system. The chromatographic device comprises a chromatographic column. The anion exchange chromatography main unit comprises 1 or more sub-units, which is implemented by running one or more chromatography columns in flow-through mode for 1 or more cycles. The management system commands an actuator (e.g., a pump or valve) to move or stop the flow of material in the flow path based on the operation of the adjacent step, the binding status of the ion exchange chromatography medium, and a pre-set algorithm.

Example 6 Virus removal Master Unit 2

The virus removal main unit 2 is a virus removal filtering system and comprises a constant pressure pump and a virus removal filter, wherein one inlet of the constant pressure pump is connected with a buffer volume 5(Tank5), virus removal filtering is carried out under set pressure, and a filtered sample flows out of the virus removal main unit 2.

EXAMPLE 7 concentration and/or exchange liquid Main Unit

1. Concentration and/or liquid change Main Unit 1 (unidirectional tangential flow filtration (SPTFF))

The concentration and/or liquid change main unit 1 is an ultrafiltration concentration system and consists of a constant pressure pump and a unidirectional tangential flow concentration membrane, wherein one inlet of the constant pressure pump is connected with a buffer volume 6(Tank6), concentration is carried out under a set pressure (1.5Bar), and a sample flows out of the concentration and/or liquid change main unit 1 after concentration.

After the end of the concentration, the membrane package is rinsed at a predetermined rinsing volume.

2. Concentration and/or liquid change main unit 2 (tangential flow liquid change)

The concentration and/or liquid change main unit 2 is an ultrafiltration liquid change system and consists of a pump and a unidirectional in-situ filter washing membrane package, wherein the inlet of the pump is connected with a buffer volume 7(Tank7), and unidirectional washing and filtration are carried out under the condition of program setting.

The peristaltic pump is used as an actuator for regulating the flow or stopping of the material flow. The pressure at the inlet of the tangential flow liquid exchange unit is controlled to be constant (15-25 psi). The ratio of the rotational speeds of the feed peristaltic pump and the displacement peristaltic pump is adjusted in a constant ratio (about 4-5 times).

After the liquid change is finished, the membrane package is washed by a preset washing volume.

And the sample is the final product solution after the solution is changed.

Claims

1. A purification system for performing continuous purification comprising, in order:

and wherein one of the concentration and/or exchange main units is an ultrafiltration concentration system comprising a pump and a tangential flow concentration membrane module, preferably a unidirectional tangential flow concentration membrane module;

2. Purification system for continuous purification according to claim 1, the control of the purification system being jointly controlled by a weight sensor and/or a level sensor,

(1) when the feedback value of the weight sensor and/or the feedback value of the liquid level sensor of the buffer volume at the upstream of the main affinity chromatography unit reaches a preset value, the purification system is started;

3. The purification system for continuous purification according to claim 1 or 2, wherein the management system allows the at least one virus-inactivation subunit of the at least one virus-inactivation master unit to start operation after the target feed liquid of the at least one affinity chromatography subunit of the at least one affinity chromatography master unit flows out of the affinity chromatography master unit; after the target feed liquid of at least one virus inactivation subunit of at least one virus inactivation main unit flows out of the virus inactivation main unit, the management system allows at least one concentration and/or liquid change subunit of at least one concentration and/or liquid change main unit to start operation, and after the target feed liquid of the first subunit of the affinity chromatography main unit flows out of the affinity chromatography main unit, the virus inactivation main unit and the operation time of the affinity chromatography main unit at least partially overlap; the management system allows the operation time of the virus inactivation main unit and the concentration and/or liquid change main unit to at least partially overlap after the first subunit target liquid of the virus inactivation main unit flows out of the virus inactivation main unit.

4. The purification system for carrying out continuous purification according to any one of claims 1 to 3, further comprising, between the affinity chromatography main unit and the concentration and/or exchange liquid main unit, at least one cation exchange chromatography main unit and/or at least one anion exchange chromatography main unit, the cation exchange chromatography main units each independently comprising one or more cation exchange chromatography sub units, and the anion exchange chromatography main units each independently comprising one or more anion exchange chromatography sub units.

5. Purification system for carrying out continuous purification according to any one of claims 1 to 4, comprising in succession:

6. Purification system for performing continuous purification according to any one of claims 1 to 5, wherein said management system allows at least one subunit of at least one upstream main unit immediately downstream of said upstream main unit to start operation after at least one subunit target feed liquid of at least one upstream main unit has flowed out of said main unit; and said management system allows said upstream master cell to overlap at least a portion of its immediate downstream master cell operating time after said upstream master cell has had its first subunit target feed solution flowing out of said upstream master cell.

7. Purification system for carrying out continuous purification according to any one of claims 1 to 5, said continuous purification being a continuous, closed, automated process for the isolation and purification of target molecules.

8. A continuous purification process for a target molecule, comprising the steps of flowing a clarified stock solution comprising the target molecule into a buffer volume immediately upstream of at least one affinity chromatography master unit as defined in any one of claims 1 to 7, and isolating and purifying the clarified stock solution by a purification system as defined in any one of claims 1 to 7.