Modular device and method for continuously producing biotechnological products

Abstract

The present invention relates to a device for continuously purifying a biotechnological product, wherein the device has a modular structure with individual modules connected to one another, wherein the modules comprise at least one tandem separation module, at least one pump module and at least one metering module, characterised in that the device is designed such that an uninterrupted continuous product flow is possible from the first to the last module. The invention also relates to a method for continuously purifying a biotechnological product in a device having a module structure with individual modules connected to one another, wherein the modules comprise at least one tandem separation module, at least one pump module and at least one metering module.

Description

[0001] MODULAR DEVICE AND METHOD FOR THE CONTINUOUS PRODUCTION OF BIOTECHNOLOGICAL PRODUCTS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a device for the continuous purification of biotechnological products, wherein the device has a modular structure with individual modules connected to one another.

[0004] BACKGROUND OF THE INVENTION

[0005] A process for the production of biopharmaceutical and biological products typically includes a perfusion culture and cell retention system upstream in the case of continuous processing or a fed-batch culture in the case of semi-continuous processing.

[0006] Downstream, such a process typically includes the steps of cell separation or the separation of cell debris, buffer or media exchange (preferably with concentration), bioburden reduction (preferably with a sterile filter), and capture chromatography. Further steps are typically performed to purify the product stream, in particular virus inactivation, neutralization, and optionally further bioburden reduction (with a sterile filter). Given the high quality standards in the production of biopharmaceuticals, additional steps typically follow: chromatographic intermediate and fine purification, bioburden reduction (e.g., with a sterile filter), virus filtration, buffer exchange, and preferably concentration.

[0007] To ensure high purity in the protein purification of monoclonal antibodies (mAbs), three consecutive chromatography steps are typically performed. In the first purification step, the mAb is isolated from the contaminated supernatant in the highest possible yield (capture). State-of-the-art purification is via affinity chromatography. The antibodies have a region that specifically binds to Protein A. Protein A chromatography achieves high yields (>90%) and removes most of the contaminants, such as host cell proteins (HCPs) and DNA. This step also typically involves the initial virus inactivation. Elution is achieved by reducing the pH (<3). Before the eluate is neutralized again, it is kept at a low pH for an extended period to inactivate the virus.

[0008] Due to the typically large batch volume (>2000 L), large-volume chromatography columns (diameter > 1 m) are also required. The chromatography gel accounts for a significant portion of the production costs. The use of Protein A can also be problematic for product quality. Protein A is also an important aspect from the perspective of the required quality attributes. It can dissolve from the material and is then found in the eluate.

[0009] In the field of chromatography, efforts are being made to ensure continuous process control. Well-known methods for this include simulated moving bed chromatography (SMBC) and periodic countercurrent chromatography (PCCC). These methods mimic continuous process control by connecting multiple columns in series. The product flow always remains discontinuous.

[0010] WO 2012 / 078677 describes a method and a system for the continuous production of biopharmaceutical products by chromatography and their integration into a production facility, particularly a single-use facility. In particular, WO 2012 / 078677 describes the use of containers (=bags) between units connected in series.

[0011] WO 2014 / 137903 describes a solution for the integrated continuous production of a protein substance in a production plant comprising columns for carrying out the production steps, which are connected in series. WO 2014 / 137903 discloses that the product flow in the continuous process is ideally controlled so that, if possible, each step or unit runs simultaneously at a similar feed rate to minimize production time. WO 2014 / 137903 discloses the use of containers between successive units that can hold the product flow for a certain period of time.

[0012] EP 3 093 335 A1 describes a modular production plant for the continuous manufacture or processing of biopharmaceutical products and a computer-implemented method for process control of the modular plant. According to EP 3 093 335 A1, a module or unit can only be operated semi-continuously if operating the module requires changing an element to perform the step.

[0013] Disadvantages of the known continuous or semi-continuous systems are that they can only guarantee short process runtimes of a few hours. The systems generally cannot be opened and returned to their original state during the process and therefore do not allow for in-process maintenance. They are either non-sterile multi-use systems or fully sterilizable single-use systems (Agilitech).

[0014] SUMMARY OF THE INVENTION

[0015] The present invention is based, among other things, on the development of tandem separation modules suitable for use in a device with a truly continuous product flow, which can be operated and maintained without interrupting the mass flow. Furthermore, the invention is based on suitable control and monitoring concepts for fully automated operation, which, for example, control the product flow via pump modules according to the invention and ensure a continuous product flow without the use of containers between successive modules.Consequently, according to a first aspect, the invention relates to a device for the continuous purification of a biotechnological product, wherein the device has a modular structure with individual modules connected to one another, wherein the modules comprise at least one tandem separation module, at least one pump module, and at least one dosing module, characterized in that the device is designed such that an uninterrupted continuous product flow is enabled from the first to the last module.

[0016] According to a second aspect, the invention relates to a tandem separation module for use in a process for the continuous purification of a biotechnological product, characterized in that the tandem separation module comprises two identical flow separation units, selected from filters and chromatography columns, connected in parallel, wherein the two flow separation units are alternately connectable to the product stream, and wherein the tandem separation module has at least one pressure sensor connected to the control of the module, and is particularly preferably designed to be sterilizable.

[0017] An optionally included sampling module according to the invention enables sampling without interrupting the mass flow.

[0018] Consequently, according to a third aspect, the invention relates to a sampling module for use in a method for the continuous purification of a biotechnological product, which is designed to take a sample from a continuous system without interrupting the product flow, wherein the sampling module preferably comprises a sample receiving container and a buffer container, which are arranged to be simultaneously connectable to the product flow, so that liquid can flow from the product flow into the sample receiving container and buffer from the buffer container into the product flow simultaneously and to the same extent.

[0019] To design the device, the individual modules are preferably combined to form devices that are suitable for carrying out the individual steps of the purification process, for example a DNA separation device, a protein separation device, a concentration device, or a buffering device, a resolubilization device.

[0020] According to a fourth aspect, the invention relates to a method for the continuous purification of a biotechnological product in a device having a modular structure with individual interconnected modules, wherein the modules comprise at least one tandem separation module, at least one pump module, and at least one dosing module, comprising the following steps:

[0021] - continuously feeding a product stream containing the biotechnological product and impurities to a first purification device of the apparatus, the device comprising at least one tandem separation module, optionally at least one dosing module and optionally a mixing module;

[0022] - uninterrupted continuous transport of the product stream from one module of the purification facility to the next,

[0023] - Continuous discharge of the product stream with reduced contaminants from the facility.

[0024] FIGURES

[0025] Fig. 1 shows schematic representations of the tandem separation modules according to the invention: A) a tandem tangential flow filtration (TFF) module comprising two TFFs connected in parallel; B) a tandem dead-end filter module comprising two dead-end filters connected in parallel.

[0026] Fig. 2 shows a schematic representation of the inventive

[0027] Dosing module.

[0028] Fig. 3 shows a schematic representation of the inventive

[0029] Sampling module. Fig. 4 shows a schematic representation of the mixing module according to the invention.

[0030] Fig. 5 shows a flow diagram of an apparatus according to the invention for producing and purifying antibodies. The individual modules are named, and modules of the same type are numbered consecutively in the order in which they are used in the process. Identical numbers for pumps indicate their membership in a common pump module.

[0031] Fig. 6 shows a schematic representation of a device according to the invention for the production and purification of antibodies, divided into sections AF for the sake of clarity. The device comprises, connected to the bioreactor, a cell separation device containing a tandem TFF module (Fig. 6 A); a DNA separation device containing a dosing module (Fig. 6 A), a mixing module, a tandem dead-end module and a sampling module (Fig. 6 B); a protein separation device containing a dosing module, two mixing modules and a tandem TFF module (Fig. 6 C), a concentration and

[0032] Buffer transfer device containing a sampling module, a mixing module, and a tandem TFF module (Fig. 6 D); a virus inactivation device containing a dosing module and a mixing module (Fig. 6 E); buffer transfer device containing a mixing module (Fig. 6 F). The numbers on the pumps indicate their affiliation to a pump module. The inlets and outlets of the individual modules are marked, as are the positions of the flow and pressure sensors.

[0033] Fig. 7 shows a flow diagram of an inventive device for solubilizing and purifying inclusion bodies. The individual modules are named, and modules of the same type are numbered consecutively in the order in which they are used in the process. Identical numbers for pumps indicate their membership in a common pump module.

[0034] Fig. 8 shows a photograph of an assembled sampling system with all connections. Components and connections are labeled in the figure.

[0035] Fig. 9 shows the transmembrane and axial pressure of two interconnected TFF modules throughout the process run. A pressure increase indicates membrane fouling. Switching to the second filter leads to a pressure drop. Six switchovers occurred in the first stage and two in the second stage.

[0036] Fig. 10 shows the process evolution for CaCl2 and PEGeooo precipitation steps. A) Concentration of soluble hcDNA at increasing CaCl2 concentrations (0-350 mM). B, C, D) Yield of trastuzumab precipitation, concentration of soluble hcDNA, concentration of soluble HCPs with (white circles, dashed line) or without (black circles, solid line) CaCO2 pretreatment of the cell culture supernatant at increasing concentrations of PEGeooo (0-15% w / w). The error bars represent the standard deviation of triplicate determinations.

[0037] Fig. 11 shows screening and testing of depth filters for the removal of precipitated hcDNA after adding a final concentration of 100 mM CaCl to the cell culture supernatant. A) Screening of depth filters at a volumetric flow rate of 250 L / m2. B) Long-term testing of the Ertelalsop B4E7 depth filter for use in a continuous process. Both screening and long-term testing were conducted at a flow rate of 1.7 mL / min (40 LMH).

[0038] Figure 12 shows critical flux experiments of hollow fiber modules conducted in recycling mode at a shear rate of 1600 s-1 and a precipitated monoclonal antibody concentration of 5 g / L in 18% PEG, 100 mM MOPS buffer, pH 7.0. The pore size and inner diameter (ID) for each module are given in the figure legend, as is the maximum critical flux (LMH). Modules 1, 2, and 3 had a path length of 30 cm. Only module 4 had a path length of 20 cm.

[0039] Figure 13 shows a flow diagram of the integrated perfusion capture setup. The clarified cell culture supernatant from the perfusion bioreactor cell retention device (TFF) undergoes two consecutive precipitation steps (CaCO and PEG), separated by a depth filtration step. The precipitated antibody is concentrated in the first TFF stage and passed to the second TFF stage, where it is washed by adding fresh buffer, reconcentrated, and collected as a precipitate.

[0040] Figure 14 shows the concentration profile of trastuzumab. Black circles, solid line, trastuzumab concentration profile in the effluent at the exit of the deposition unit; black triangles, solid line, and white triangles, dashed line, trastuzumab concentration profile in the permeate from stage 1 and stage 2 of the TFF, respectively; blue circles, blue dashed line, trastuzumab concentration profile at the exit of the deposition unit, calculated using mass balance based on the perfusion titer and losses in the TFF permeate. A) Stand-alone deposition, B) Integrated perfusion deposition.

[0041] Figure 15 shows the determination of aggregates and monomers (%), based on the total area, obtained by analytical size-exclusion chromatography for cell culture supernatant and redissolved trastuzumab samples obtained during the processes, incubated at low pH for 1 hour (viral inactivation). A) Aggregates and monomers (%) for standalone capture over seven days of continuous processing. B) Aggregates and monomers (%) for integrated perfusion capture over eight days of processing. C) Aggregates and monomers (%) for cell culture supernatant samples from the perfusion process over eight days of process integration for integrated perfusion capture.

[0042] Fig. 16 shows the concentration (ppm) of hcDNA (A) and HCPs (B) for the cell culture supernatant used as starting material for stand-alone capture (white triangle, dashed line), for the redissolved trastuzumab obtained from the stand-alone capture (white circles, dashed line), for the cell culture supernatant from the perfusion process integrated with the capture unit (black triangles, solid line), for the redissolved trastuzumab obtained from the integrated perfusion capture (black circles, solid line).

[0043] Figure 17 shows the cumulative distribution function of the capture unit obtained by salt pulse injection (tubular reactor for antibody precipitation and two stages of TFF). Black solid line, experimental data; orange dashed line, yellow dashed line, black dashed line, theoretical cumulative distribution function obtained by applying the tank-in-series model for a series of CSTRs of 1, 5, and 10 CSTRs, respectively.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] According to a first aspect, the invention relates to a device for the continuous purification of a biotechnological product, wherein the device has a modular structure with individual, interconnected modules, wherein the modules comprise at least one tandem separation module, at least one pump module, and at least one dosing module, characterized in that the device is designed such that an uninterrupted, continuous product flow is enabled from the first to the last module. The term "product flow" in the context of the invention refers to the fluid from a heterogeneous cell culture fluid mixture containing the product, as well as the result of any other process steps of the process according to the invention, i.e., the fluid flowing between the individual modules of the process according to the invention, wherein these product flows may have different concentrations and degrees of purity.

[0046] For the purposes of the invention, a "continuous process" is understood to mean a process for carrying out at least two consecutive process steps in which the output stream of an upstream step is fed to a downstream step. The downstream step begins processing the product stream before the upstream step is completed. Typically, in a continuous process, a portion of the product stream is always conveyed in the production line and is referred to as the "continuous product stream."

[0047] The term "closed" in the context of the invention refers to the operation of the process according to the invention and the modular system according to the invention, which can be operated in such a way that the product produced and / or processed using this process and this modular system is not exposed to the ambient environment. Materials, objects, buffers, and the like can be added from outside to the closed process according to the invention and the corresponding closed modular system according to the invention, but this addition is carried out in such a way that exposure of the produced and / or processed product to the ambient environment is avoided.

[0048] According to the invention, the device is divided into modules. Each of these modules has its own process-related functions. Individual modules can be exchanged, added, or their sequence changed. Connecting lines are provided between the various modules within the device. The modules can be connected together in a sterile manner. The modules can preferably be connected by welding or using aseptic connectors such as Steam Thru Connectors® or Opta® SFT sterile connectors.

[0049] This uninterrupted continuous product flow is achieved by a suitable combination of modules and a control and regulation of the flow rate of the product stream from one module to the next.

[0050] According to one embodiment, the device is designed to be self-regulating with regard to automatically achieving and maintaining a flow equilibrium of the product flow and / or with regard to pressure distribution within the device. According to one embodiment, the device is configured such that functional elements of the modules can be serviced or replaced during ongoing operation without interrupting the product flow.

[0051] To automate the device, all pump motors can be matched to each other and controlled by manual setpoint input. For this purpose, one or more pumps are combined in a pump module.

[0052] According to one embodiment of the device, the pump module comprises one or more pumps and volume flow sensors that are connected to the pump module's control system and ensure constant flow rates. Preferably, each pump is connected to an associated volume flow sensor. The volume flow sensors are particularly preferably ultrasonic volume flow sensors. In particular, they are non-invasive clamp-on ultrasonic sensors, i.e., ultrasonic sensors that are clamped to the outside of the pipe, thus requiring no intervention in the product flow. According to one embodiment, the pump module comprises a frame with mounts for the individual pumps and the volume flow sensors.

[0053] The task of the pump module is to ensure constant flow rates over a long process period. The pumps of a pump module can be matched to each other by setting simple linear relationships between the pumps with respect to the pumping rate (P) or the generated volumetric flow rate.

[0054] E.g.: P1 = P2+P3 or P1 = x*P

[0055] A feedback loop adjusts the pump speed to the desired flow rate if necessary. The pumps used can be magnetically coupled or peristaltic pumps. The pumps of a pump module are assigned to different parts of the device. Each pump has an inlet and an outlet, which are connected to the assigned modules or connecting lines via aseptic connectors.

[0056] The set flow rates or flow velocities vary regularly from process step to process step in order to achieve the technical / economic optimum for each process step. The flow rate can range from 0.5 ml / min to 100 ml / min. For example, the flow rate can be set at 0.5 ml / min, 1.0 ml / min, 1.5 ml / min, 2.0 ml / min, 2.5 ml / min, 3.0 ml / min, 4.0 ml / min, 5.0 ml / min, 6.0 ml / min, 7.0 ml / min, 8.0 ml / min, 9.0 ml / min, 10.0 ml / min, 11.0 ml / min, 12.0 ml / min, 13.0 ml / min, 14.0 ml / min, 15.0 ml / min, 16.0 ml / min, 17.0 ml / min, 18.0 ml / min, 19.0 ml / min, 20.0 ml / min, 25.0 ml / min, 30.0 ml / min, 35.0 ml / min, 40.0 ml / min, 45.0 ml / min, 50.0 ml / min, 55.0 ml / min, 60.0 ml / min, 65.0 ml / min, 70.0 ml / min, 75.0 ml / min, 80.0 ml / min, 85.0 ml / min, 90.0 ml / min, 95.0 ml / min, or 100 ml / min. According to one embodiment, the flow rate is

[0057] process in the range of 1.0 ml / min to 80 ml / min. According to one embodiment, the flow rate is throughout the entire

[0058] process in the range of 2.0 ml / min to 60 ml / min.

[0059] According to one embodiment, the modules are controlled individually and independently of each other. In this case, there is no communication between the modules to prevent error propagation or incorrect control. Alternatively, it is also possible to link the control system to an Advanced Process Control System. This allows for process optimization and can, for example, enable model predictive control, soft sensor technology, and advanced real-time spectroscopy methods.

[0060] In its application, closed-loop control involves measuring the variable to be influenced (controlled variable) and continuously comparing it with the desired value (setpoint). Based on the deviation, a controller calculates a manipulated variable that affects the controlled variable in such a way that the deviation is minimized and the controlled variable assumes a desired time response. This corresponds to a closed-loop control process.

[0061] In comparison, control refers to the process in a system in which one or more input variables influence the output variables due to inherent system laws. Control is characterized by an open effect path or a closed effect path, in which the output variables influenced by the input variables do not constantly influence themselves and do not repeatedly influence each other via the same input variables.

[0062] The individual modules can be coupled to each other directly or indirectly via a suitable coupling element, preferably sterile.

[0063] According to one embodiment, the tandem separation module comprises two identical flow separation units connected in parallel, wherein the two flow separation units can be alternately connected to the product stream.

[0064] The flow separation units are selected from filters and chromatography columns. Specifically, they are tandem tangential flow filters (TFFs) or tandem dead-end filters. Accordingly, the modules are referred to as tandem TFF modules or tandem dead-end filter modules. In tangential flow filtration, the suspension to be filtered is pumped parallel to a membrane or filter medium, and the permeate (also called filtrate) is withdrawn perpendicular to the flow direction. The shear forces occurring on the filter surface due to the turbulent flow can be varied depending on the volume flow. The high velocity largely prevents a filter cake (cover layer or fouling) of the solid particles to be separated from building up on the membrane. This would increase the filtration resistance and thus the pressure drop across the filter, reducing the filter performance.

[0065] While in a dead-end filter, the solids to be separated are collected as a filter cake, in cross-flow filtration, the solids can only be concentrated to the point where the suspension is still pumpable. The recirculated portion of the liquid flow is called the retentate.

[0066] Alternatively, chromatography columns can be used as flow-through separation units. These columns contain a chromatography resin with functional groups that do not bind to the desired biotechnological product. Instead, the chromatography columns should contain resins that bind to the impurities in the product stream that are to be concentrated.

[0067] It is important that the tandem separation modules do not contain chromatography columns that require an elution step, as otherwise continuous processing would not be possible. In particular, the entire device does not contain any separation modules with an elution step.

[0068] The tandem separation module can contain at least one pressure sensor. Pressure sensors are preferably connected to the control system of the device or module. The pressure sensor can be used to measure the loading of the filter or chromatography column.

[0069] The tandem separation module is designed to ensure a sterile parallel connection of the flow separation units (filter or chromatography). The module can be made of stainless steel and thus steamed at 121 °C and 1.1 bar. The module preferably includes a Sterilization-In-Place (SIP) system. This ensures that the module is fully sterilized. As soon as a flow separation unit needs to be replaced, the control system can automatically switch to the second unit. The first flow separation unit can be washed and removed. A pre-sterilized filter or chromatography column, if required, is then inserted into the first as a replacement.

[0070] A flow separation unit is used. A subsequent sterilization step can be performed in which the fluid lines open during the exchange are sterilized and, optionally, the flow separation unit is also sterilized. The unit can then be filled with buffer and switched to the main flow if the parallel unit of the module needs to be replaced.

[0071] The tandem separation module can be equipped with a temperature sensor to monitor sterilization. The valves are controlled automatically.

[0072] The tandem dead-end filter module is shown in Fig. 1 B. It is the least complex tandem separation module, as it does not include a retentate outlet, and monitoring and control of the system are straightforward. The system can be monitored using pressure sensors, i.e., the inlet pressure pF or the differential pressure ΔpF = pF / PR. If the pressure rises above a threshold, e.g., 2 bar inlet pressure, the control system can automatically switch to the parallel unit.

[0073] The tandem chromatography module is designed similarly to the tandem dead-end module. It also features pressure monitoring, preferably differential pressure monitoring.

[0074] The most complex tandem separation module is the tandem TFF module (Fig. 1A). According to one embodiment, the tandem TFF module comprises a filter holder, preferably with adjustable clamps, for example, with a length in the range of 30–65 cm and a diameter in the range of ~0.5–5 cm. The tandem TFF module features either a Clean-In-Place (CIP) system or a SIP system. The tandem TFF module features at least three pressure sensors: for the inlet, the retentate, and the permeate. In a SIP system, at least one additional pressure sensor is required for the steam inlet.

[0075] Tangential filters are preferably hollow fiber filters or membranes. These can have an area of ​​around 100 cm 2 - 900 cm 2 The tangential filters have one inlet and two outlets for permeate and retentate.

[0076] Due to the existing retentate outlet, more valves are required, and a separation of the two permeate outlets may be necessary (due to the sterilization process). The transmembrane pressure (TMP) serves as the control variable. This is derived from the measured pressures of the inlet, retentate, and permeate.

[0077] TMP = (pt + p r ) / 2 +p P

[0078] PF = inlet pressure pp = permeate pressure PR = retentate pressure

[0079] Alternatively, the differential pressure, i.e. the quotient of the inlet pressure and the retentate pressure (PR), Ap = pt / PR, can also be the control variable. The threshold for the transmembrane pressure (TMP) is up to 2.5 bar, and for the differential pressure, it is below 2 bar.

[0080] According to a second aspect, the invention provides a tandem separation module for use in a process for the continuous purification of a biotechnological product, comprising two identical flow-through separation units, selected from filters and chromatography columns, connected in parallel. The two flow-through separation units can be alternately connected to the product stream. The tandem separation module includes at least one pressure sensor connected to the module's control system and is particularly preferably designed to be sterilizable. The tandem separation module according to the second aspect is particularly as described in the first aspect.

[0081] According to one embodiment, the dosing module is designed to ensure continuous dosing of additives. The dosing module can comprise a supply container and an intermediate container. A filter unit is preferably arranged between these containers. This filter unit is advantageously equipped with a pressure sensor.

[0082] The task of the dosing module (Fig. 2) is to ensure the continuous dosing of buffers and other solutions (e.g. precipitant) over a long period of time. The module has a large supply container with a volume of at least 40 l, preferably at least 50 l, particularly preferably at least 60 l, and a smaller intermediate container of less than 1 l, preferably less than 800 ml, particularly preferably less than 500 ml. Both the supply container and the intermediate container are preferably bags. The intermediate container ensures that there are no interruptions in the mass flow when the supply container is replaced. To maintain the sterility of the system, a dead-end filter capsule can be installed upstream of the intermediate container and monitored by a pressure sensor. The supply container can be monitored gravimetrically.

[0083] In addition to the at least one tandem separation module, at least one pump module, and at least one dosing module, the device preferably has at least one sampling module and / or a mixing module.

[0084] According to one embodiment, the mixing module is designed to mix several partial streams with a narrow residence time distribution. The average residence time is calculated as the quotient of the fluid volume in the reactor to the exiting volume flow. The average residence time is a measure of the average time required by the reaction mass to flow through a reactor. However, the effective residence times of individual particles can vary greatly, resulting in a residence time distribution.

[0085] Depending on the reaction to be carried out, a suitable residence time for the corresponding mixing module can be defined by dimensioning the mixing module and setting the flow rate. In the process according to Example 1, the average residence times are in the range of 5 min to 60 min. For homogeneous conversion or mixing, the variance in the residence time should be kept as low as possible. The mixing module preferably has a tubular reactor. Even at a low flow rate of less than 3 ml / min, the variance in the residence time of a tubular reactor with a volume of 2 L is only about half that of a stirred tank with the same volume. Furthermore, at higher flow rates, the variance in the residence time can be reduced even further. At a flow rate of 7.5 ml / min, the variance is only half that of approximately 3 ml / min.The tubular reactor is, in particular, a tubular system with a mixing insert. The mixing module is preferably disposable.

[0086] The term "disposable article" in the context of the invention means that the corresponding product-contacting parts, in particular apparatus, containers, filters, and connecting elements, are suitable for single use with subsequent disposal. These containers can be made of plastic or metal. Within the context of the invention, the term also includes reusable articles, for example, made of steel, which are used only once in the process according to the invention and are then no longer used in the process. Such used disposable articles can then also be referred to as "disposable" or "single-use" articles in the process according to the invention ("SU technology").

[0087] According to one embodiment, the mixing module has a spectrometer for determining the volumetric concentration and / or the mixing efficiency. The mixing module (Fig. 3) thus serves to optimally mix multiple streams homogeneously with the shortest possible residence time distribution. It is possible to connect up to two flow cells to measure the mixing efficiency, but also the volumetric concentration (and thus also the protein concentration) using a spectrometric method. The mixing module preferably has no control algorithms implemented. The tubular reactor can consist of a hose system with a mixing insert. The mixing module is preferably a disposable item. The mixing module has at least two inlets and at least one outlet, preferably two outlets.

[0088] According to one embodiment, the sampling module is designed to take a sample from the continuous system without interrupting the product flow. According to one embodiment, the sampling module comprises a sample receiving container and a buffer container, which are arranged to be simultaneously connectable to the product flow, so that liquid can flow from the product flow into the sample receiving container and buffer can flow from the buffer container into the product flow at the same time.

[0089] The task of the sampling module is to take a sample from a continuous system without interrupting the mass flow. During sampling, the product flow is diverted away from the process direction into a sampling vessel. Simultaneously, the path to a buffer tank opens, which replaces the missing volume flow in the main line in the process direction. After sampling, the valve returns to the inlet position, and the product flow returns to the next module in the process direction. The fill level of the buffer tank can be monitored gravimetrically. The buffer flow rate can be monitored via an ultrasonic clamp-on sensor, and the pump can be controlled via a feedback loop. According to a third aspect, the invention provides a sampling module as described above.

[0090] According to one embodiment, the device is designed to perform multiple purification steps. For this purpose, the device preferably comprises several devices constructed from the modules.

[0091] An example of such a device is a cell disruption or cell separation device. This contains, in particular, a dosing module, a mixing module, and a tandem separation module. An example of such a device is a DNA separation device. This contains, in particular, a dosing module, a mixing module, and a tandem separation module. Another example of such a device is a protein separation device. This contains, in particular, a dosing module, a mixing module, and a tandem separation module. Another example of such a device is a concentration device. This contains, in particular, a tandem separation module. Another example of such a device is a buffer re-adjustment device. This contains, in particular, a dosing module, a mixing module, and a tandem separation module. Another example of such a device is a resolubilization device.This device contains, in particular, a dosing module and a mixing module. Another example of such a device is a virus inactivation device. This device contains, in particular, a dosing module and a mixing module.

[0092] Due to its modular design, the device can be easily adapted for the purification of any type of protein or nucleic acid. For example, as shown in Example 2, the setup can be easily adapted to the purification of proteins from inclusion bodies. Similarly, more protein purification units can be used as needed to achieve higher purity.

[0093] The device preferably contains no intermediate tanks between the modules and / or devices. Eliminating the need for intermediate tanks is achieved by continuous operation without interruption for material exchange, the elimination of purification modules that require an elution step, and appropriately matching flow rates in the individual process sections.

[0094] According to a fourth aspect, the invention relates to a method for the continuous purification of a biotechnological product in a device having a modular structure with individual interconnected modules, wherein the modules comprise at least one tandem separation module, at least one pump module, and at least one dosing module, comprising the following steps:

[0095] - continuously feeding a product stream containing the biotechnological product and impurities to a first purification device of the apparatus, the device comprising at least one tandem separation module, optionally at least one dosing module and optionally a mixing module;

[0096] - uninterrupted continuous transport of the product stream from one module of the purification device to the next, and

[0097] - Continuous discharge of the product stream with reduced contaminants from the facility.

[0098] According to one embodiment of the method, the biotechnological product is a protein or a nucleic acid. The protein can be selected from a blood coagulation factor, a peptide hormone, a growth factor, an interferon, an interleukin analogue, or a vaccine, or an antibody, preferably antibodies of the classes IgG1-4, IgM and IgG, IgE, or derivatives thereof. The protein can also be a protein for use as food. The nucleic acid can be selected from DNA, RNA, such as antisense RNA, or antisense oligonucleotides.

[0099] According to one embodiment, the process is implemented without storing the product stream in intermediate tanks and / or without elution chromatography. According to one embodiment of the process, the device is a device according to the first aspect. According to one embodiment, the purification device uses flow-through chromatography or protein precipitation followed by filtration, for example, dead-end or tangential flow filtration.

[0100] According to one embodiment, the tandem separation module comprises two identical flow-through separation units, selected from a filter and chromatography columns, connected in parallel and a pressure sensor. One of the two flow-through separation units is connected to the product stream, and when a pressure threshold is exceeded as a measure of the flow-through separation unit's filling capacity, the product stream is switched to the other flow-through separation unit. This switchover can occur automatically or be dependent on confirmation from the system operator. In a tandem TFF module, the transmembrane pressure (TMP), the differential pressure Ap = pt / PR, and / or the pressure rise are determined. In the tandem dead-end module, the TMP threshold is in the range of 2 to 2.5 bar. The differential pressure threshold is below 2 bar.

[0101] Tandem operation allows for continuous switching between units (depth filters, membranes, etc.). Pressure monitoring prevents exceeding the critical pressure, thus enabling continuous operation (see also Figure 9). At elevated pressures, the mass flow is directed to the second stage, creating a pressure drop. The process is not stopped and continues continuously. The equilibrium state is maintained.

[0102] There is no specific threshold for the pressure increase; rather, an exponential increase, for example, signals that the filling capacity has been reached. As an additional safety feature, an alarm signal can usually be triggered even below the threshold. The first flow separation unit can then be replaced with a new one, while the connectors are sterilized.

[0103] According to one embodiment of the method, additives, such as the precipitant for precipitation, are fed into the product stream via a dosing module. The dosing module comprises a supply tank and an intermediate tank, between which a filter unit equipped with a pressure sensor is arranged. The intermediate tank is connected to the product stream, and simultaneously, the supply tank is disconnected from the product stream when the supply tank is empty and / or the pressure at the filter unit exceeds a threshold. Pressure at the filter unit above the threshold indicates contamination of the dosing module.

[0104] According to one embodiment of the method, a sample is taken from the product stream via a sampling module comprising a sample receiving container and a buffer container, without interrupting the product flow. The sample receiving container and the buffer container are simultaneously connected to the product stream, so that liquid flows simultaneously from the product stream into the sample receiving container, and buffer flows simultaneously from the buffer container into the product stream.

[0105] The main flow can be controlled via a custom-designed cylinder valve, observing the 3D rule. To seal the sampling module, an O-ring (D = 2 mm) and a pin are integrated into the cylinder valve, and polytetrafluoroethylene (PTFE) gaskets are inserted between the Tri-Clamps.

[0106] The sensors monitor the pressure. If the pressure exceeds the maximum permissible pressure, this is detected by the pressure sensors. If the sampling module is used in a plant process, an alarm would be triggered if the pressure is exceeded. This sampling loop is suitable for a 2 ml sample. The advantage of this design is that the sample volume can be adjusted by replacing the sampling loop with a longer one.

[0107] In a further embodiment of the process according to the invention, all product-contacting units or elements used in modules are germ-reduced. The term "germ-reduced" in the context of the invention refers to a state of reduced germ count, i.e., a number of microorganisms per unit area or volume of almost zero. The germ reduction method can preferably be selected from the group consisting of gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment, ozone treatment (O3), hydrogen peroxide treatment (H2O2), and steam-in-place (SIP) treatment.

[0108] According to one embodiment of the method, the purification comprises one or more of the following steps in suitable devices: cell separation, DNA separation, protein separation, buffer transfer, protein concentration and virus inactivation. In a preferred embodiment, the biotechnological product is an antibody and the method comprises each of the mentioned steps. According to one embodiment of the antibody purification, the flow rate in the cell separation device is in the range of 5 ml / min to 10 ml / min, preferably in the range of 6 ml / min to 9 ml / min, particularly preferably in the range of 6.5 ml / min to 7.5 ml / min. According to one embodiment, the flow rate in the DNA separation device is in the range of 5 ml / min to 10 ml / min, preferably in the range of 6 ml / min to 9 ml / min, particularly preferably in the range of 7 ml / min to 8 ml / min.According to one embodiment, the flow rate in the protein separation device is in the range from 7 ml / min to 14 ml / min, preferably in the range from 8 ml / min to 12 ml / min, particularly preferably in the range from 9 ml / min to 11 ml / min. According to one embodiment, the flow rate in the buffer recirculation device, the concentration device and the virus inactivation device is in the range from 0.5 ml / min to 5.0 ml / min, preferably in the range from 1.0 ml / min to 3.0 ml / min, particularly preferably in the range from 1.0 ml / min to 2.0 ml / min. According to one embodiment of the method, the flow equilibrium of the continuous purification is maintained over a period of at least one week, preferably at least 4 weeks, particularly preferably at least 3 months, preferably at least 6 months.Such a long running time of several weeks of continuous operation is made possible by the closed and germ-reduced operation of the device, as well as the possibility of maintenance of the individual modules and material exchange during operation, for example through the use of tandem separation modules with flow units.

[0109] According to one embodiment of the process, the continuous purification is coupled with continuous protein production. Continuous protein production can be carried out in a continuously operated bioreactor.

[0110] EXAMPLES

[0111] Example 1 - Purification of monoclonal antibodies (mAbs) from cell culture supernatants

[0112] mAb purification can consist of the following 6 process steps:

[0113] • Cell disruption or cell separation

[0114] • DNA separation or precipitation

[0115] • (selective) protein precipitation

[0116] • Concentration

[0117] • Wash & re-buffer

[0118] • Resolubilization

[0119] • Neutralization

[0120] A schematic modular design of the device for the continuous purification of monoclonal antibodies is shown in Fig. 6 and as a flow diagram in Fig. 5. The cell suspension obtained from the bioreactor process is first freed from the cells by filtration (cell separation device) and transferred to the DNA separation device, containing a dosing module, a tubular reactor module and a tandem dead-end module as well as pumps of a pump module.

[0121] The dosing module is used to adjust the CaCl concentration in the product stream to 100 mM from a CaCl2 stock solution. If necessary, a minimal concentration of phosphate may be added to the supernatant beforehand. The reaction time for DNA precipitation is a few minutes. Optimal mixing and the necessary residence time are achieved over the length of the tubular reactor module. The precipitate is then filtered through a depth filter (23 cm) in the tandem dead-end module. 2 up to 250 cm 2 ) is separated. The system is monitored via pressure (max. 2 - 2.5 bar), and if the filter fouls, the system automatically switches to the second filter. The filter can be replaced, the path can be steamed (121 °C, 1.1 bar), and reinserted into the system in a sterile condition (functionally closed system).

[0122] Protein precipitation takes place in a protein separation device containing a dosing module for the addition of chaotropic substances. MAb precipitation occurs at a PEGe (polyethylene glycol) concentration of 12–15 vol%. This substance is also added from an approximately 50 vol% stock solution via a dosing module. The reaction time for this precipitation is approximately 20 minutes, and, as in the first step, optimal residence time and mixing are achieved via a tubular reactor module.

[0123] Concentration (5-20 times) takes place in a rebuffering device with a tandem TFF system. Liquid is continuously removed and newly precipitated protein is added. Here, too, the system is monitored via pressure or transmembrane pressure, and in the event of increased membrane fouling (pressure increase), the system automatically switches to the second filter. The unused filter can be replaced, and the path can be steamed and reinserted into the system under sterile conditions (functionally closed system). Rebuffering or washing of the precipitate takes place in a rebuffering device analogous to concentration, except that an additional line containing an aqueous buffer (e.g., 100 mM MOPS buffer) is connected to the TFF system. A rebuffering factor of 10 is aimed for in order to free the precipitate as much as possible from the supernatant and any remaining soluble media and cell culture components.Here, too, the system is monitored via pressure and has the redundancy mentioned above.

[0124] The precipitate is dissolved in the next step. This can be done at a low pH (< 3.2). The reaction itself requires only a short residence time, but can be extended to 1 h to simultaneously perform virus inactivation. The optimal mixing and residence time are determined by the length of the tubular reactor module. In the final step, the solution is neutralized (pH = 7).

[0125] The individual solutions are added via the dosing modules. All flow rates are monitored by flow sensors and controlled by the associated pumps. The system is continuously in steady state. There are no intermediate containers that negatively impact the residence time.

[0126] Example 2 - Purification and resolubilization of inclusion bodies

[0127] Inclusion Body (IB) purification and resolubilization includes the following steps:

[0128] • IB solubilization

[0129] • IB Refolding

[0130] • Rebuffer protein solution

[0131] • Concentrate protein solution

[0132] A schematic modular structure of the device for the continuous processing of inclusion bodies is shown in Fig. 7.

[0133] IBs, also called inclusion bodies, originate from a microbial process, namely protein expression in bacteria, particularly Escherichia coli. During expression, the target protein, produced in large quantities, misfolds, aggregates, and thus forms IBs, in which the target protein is improperly folded but is highly pure. After rupturing the cell, the IBs can be separated from the cell debris and the remaining medium by centrifugation.

[0134] The IB pellet is a slurry with a high protein content. If necessary, buffer can be added to the IB slurry to adjust it to the desired final protein content.

[0135] The diluted slurry is pumped into the continuous system. In the first process step, the IBs are solubilized with a chaotropic or neutral detergent of high molarity, e.g., 8 M guanidine hydrochloride or 6 M urea. Depending on the product, if disulfide bonds need to be formed, this process must already take place under reducing conditions. This is usually done with the addition of dithiothreitol (DTT), e.g., 100 mM.

[0136] After solubilization, the protein refolds. By buffering, for example, with a 100 mM Tris-HCl buffer system with stabilizing additives such as 300 mM NaCl and 50 mM arginine at a pH of 7, the solubilized protein is folded into its active form.

[0137] In the final step, the diluted protein solution (usually) resulting from the process is concentrated.

[0138] The tandem filter modules used correspond to those in Example 1. Furthermore, the same control strategies are applied. However, due to the size of the IBs, (hollow fiber) filter membranes with a different cutoff are used. For the first tandem TFF module, where the target protein is still in the aggregated solid state, a hollow fiber membrane with a cutoff of 0.2 pm is used. From the solubilization step onwards, membranes with cutoffs of 3-10 kDa are used, depending on the size of the target protein. Theoretically, any protein that can be expressed in E. coli and exists as an IB can be continuously purified using this process setup. Examples of industrially relevant products include insulin, FAb fragments (fragments of monoclonal antibodies), and casein.

[0139] Example 3 Production of a recombinant IgG1 and purification using the continuous process according to the invention in a modular production plant

[0140] 3.1 Methods

[0141] 3.1.1 Cell culture

[0142] In this study, a recombinant IgG1 (trade name trastuzumab, PI 8.4-8.6) was used. CHO cells were cultured in perfusion mode in a Labfors 5 benchtop bioreactor (Infors Ht, Switzerland). Cell proliferation was performed in shake flask cultures (Corning, New York, USA) with a working volume of up to 500 mL. The bioreactors were operated at a cell density of 3-5 x 10 6 cells / mL. Perfusion was initiated on the same day at 0.5 WD by continuously adding fresh perfusion medium and removing harvested cells from the permeate. A perfusion rate of 0.5 WD was maintained until a cell density of 10 x 10 6cells / mL was reached and the cell density was gradually increased up to 1 WD until the target value for the viable cell concentration (VCC) of 60 ± 10 x 10 6Cells / mL was reached. Cell bleeding was applied via an automated bleeding feedback loop with VCC prediction from an online capacity probe (Incyte, Hamilton, Switzerland). The working volume was maintained at 2 L by constant inflow and outflow rates. Feedback loops of bioreactor weight, feed, and bleed were used to adjust the pump flow rates. The cultures were perfused with a medium consisting of 4Cell XtraCHO production medium (Sartorius) with Feed A and Feed B (6% / 1%) supplement (Sartorius) and 60 mM D-glucose. The setpoints for the process temperature, dissolved oxygen concentration, and culture pH were 37°C, 40%, and 7.0 ± 0.1, respectively.To collect supernatant for process development and batch capacity, supernatant was harvested from the bioreactor using an ATF 2 (Repligen, Waltham, MA) cell retention device equipped with a polyethersulfone HF with a pore size of 0.2 pm and a surface area of ​​0.13 m. 2 at a shear rate of 1500 s -1 (Repligen, Waltham, MA). For integrated perfusion detection, the supernatant was harvested over TFF using a polyvinylidene fluoride HF (Asahi Kasei, Tokyo, Japan) with a pore size of 0.2 pm and a membrane area of ​​0.08 m 2 at a shear rate of 1000 s -1 A magnetically levitated pump (Levitronix, Framingham, MA, USA) was used for recirculation.

[0143] 3.1.2 hcDNA precipitation by CaCF

[0144] To determine the best conditions for efficient hcDNA precipitation, increasing concentrations of CaCl2 from 0 to 350 mM were added to the CCCS from a 1 M stock solution of CaCl2 in 100 mM MOPS, pH 7.0. Screening was performed in 1.5 mL centrifuge tubes with a total working volume of 1 mL. Samples were gently shaken for 5 minutes on an end-to-end shaker (Stuart Rotator SB3; Cole-Parmer, Vernon Hills, IL). This time was sufficient for maximum hcDNA precipitation at the corresponding CaCl2 concentration. After precipitation, samples were centrifuged at 6000 g for 6 minutes, and the supernatant was collected in fresh centrifuge tubes for subsequent analysis.

[0145] 3.7.3 Deep filtration of precipitated impurities

[0146] Depth filter capsules with different pore sizes from two different manufacturers were tested to remove CaCl precipitated contaminants from the product stream. A portable 2100Q turbidity meter (HACH Lange GmbH, Düsseldorf, Germany) was used for turbidity measurement. A PressureMAT® with a bench-scale pressure sensor (PendoTech, New Jersey, USA) was used to record the inlet pressure. Qubicon® online software (Qubicon, Vienna, Austria) enabled online monitoring and recording of the inlet pressure during the experiments. For each depth filter tested, a final concentration of 100 mM CaCl2 from a 1 M stock solution of CaCO in 100 mM MOPS, pH 7.0, was added to the CCCS in standard laboratory glassware. The precipitate was kept in suspension using a magnetic stirrer. Depth filters were aerated at 40 L / m before use. 2deionized water. A peristaltic pump (114 DV, Watson Marlow, Guntramsdorf, Austria) was used to pump the solution at 1.7 mL / min (40 LMH) up to a volumetric load of 250 L / m 2 to the depth filter inlet. Turbidity measurements were taken every 30 minutes at the depth filter outlet and in the depth-filtered solution pool. PharMed® BPT tubing (Saint-Gobain, Courbevoie, France) was used on the pump head and connected with Masterflex® Luer Lock connectors to silicone tubing (Tygon R-3603, Saint-Gobain, Courbevoie, France) for the depth filter inlet and outlet lines. All tubing had an inner diameter (ID) of 3.2 mm (3 / 32 inch).

[0147] 3. 1.4 Trastuzumab precipitation by PEGeoooo

[0148] Increasing concentrations of PEGeooo ranging from 0 to 15% (w / w) were added to the CCCS from a 50% stock solution of PEGeooo in 100 mM MOPS pH 7.0. Alternatively, CCCS was pretreated with a final concentration of 100 mM CaCl to evaluate the effects of CaCl precipitation on trastuzumab uptake by PEGeooo. The experiments were conducted in

[0149] 1.5 mL centrifuge tubes with a working volume of 1 mL were used. After the addition of PEGeooo, the samples were incubated for 15 minutes on an end-to-end shaker (Stuart Rotator SB3; Cole-Parmer, Vernon Hills, IL). For subsequent analysis, the samples were centrifuged at 2000 g for 2 minutes, and the supernatant was collected in fresh centrifuge tubes.

[0150] 3. 1.5 Experiments on critical flow

[0151] Critical flow experiments were conducted on various HFs according to Li and Zidney (2017). The precipitate was pumped from a 100 mL reservoir at a concentration of 5 g / L to the HF inlet using a peristaltic pump (313 DV Watson Marlow, Guntramsdorf, Austria), with the retentate and permeate being recycled back into the same reservoir. A second peristaltic pump (114DV, Watson Marlow, Guntramsdorf, Austria) was used to control and modulate the permeate flow, which was varied within a range of 11 LMH to 109 LMH. The transmembrane pressure (TMP) was monitored by installing bench-scale pressure sensors (PendoTech, New Jersey, USA) on the HF inlet, retentate, and permeate lines. The pressure sensors were connected to a PressureMAT® (PendoTech, New Jersey, USA) and Qubicon® online software was used for monitoring and recording.

[0152] 3. 1.6 Resolubization of trastuzumab at low pH

[0153] Resolubization of captured trastuzumab was performed batchwise using a 50 mM phosphate buffer (pH 2.5) at a sample:buffer volumetric dilution ratio of 1:5. Samples were incubated for 60 minutes at low pH (<3.5) on an end-to-end shaker (Stuart Rotator SB3; Cole-Parmer, Vernon Hills, IL) and neutralized by adding 200 mM phosphate buffer (pH 8.0) at a sample:buffer volumetric dilution ratio of 4:1. This ratio resulted in a solution with a final pH of 6.5. Samples were then analyzed immediately, stored at 4°C, or frozen at -20°C for longer storage.

[0154] 3.1.7 Continuous 2-stage precipitation and 2-stage TFF

[0155] The supernatant harvested during a perfusion process was either collected in disposable 10 L Flexboy® 2D bags (Sartorius, Ort) and frozen at -20°C before being used for stand-alone trastuzumab precipitation or directly passed to the first precipitation step in a poolless integrated perfusion capture system. Stock solutions of CaCl and PEG were prepared according to previous experiments. All solutions were sterile filtered using 0.2 pm Sartopore® 2 XLG capsule filters (Sartorius) in previously autoclaved plastic bottles with 3-way caps (Nalgene, Rochester, USA). Peristaltic pumps were used to transport the fluids. All tubing used in the setup was silicone tubing (3.2 mm ID) or PharMed® BPT tubing (Saint-Gobain, Courbevoie, France, 3.2 mm ID) on the heads of the peristaltic pumps. The tubular reactors were manufactured in-house using static mixers (HT-40-6).30-24-AC; material: acetal; Stamixco AG, Wollerau, Switzerland) were inserted into flexible standard laboratory tubing (Tygon® R-3603, 4.8 mm inner diameter; Saint-Gobain, Courbevoie, France), which was arranged spirally and stacked vertically. The length of the tubular reactors was determined based on the residence time required for efficient precipitation (5 minutes for CaCl precipitation, 15 minutes for PEG precipitation).

[0156] 3. 1.8 Flow-through anion exchange membrane chromatography

[0157] The precipitate harvested during the integrated perfusion acquisition process was dissolved at low pH, as reported in Section 2.6. For process optimization, we applied a DoE approach. The software used for experimental design and statistical analysis was Design-Expert® (Stat-ease Inc.). The pH of the solution was neutralized to the required pH by titration with 0.02 M NaOH. The conductivity was adjusted by adding 1 M NaCl or by dilution with HQ water. For these experiments, an Äkta pure 25 (Cytiva) and a negatively charged membrane adsorber, the 3M™ Polisher ST (1 cm2 area), were used. Before loading, the device was flushed with 400 L / m2 of a 50 mM phosphate buffer, the pH and conductivity of which were adjusted according to the feed solution. 50 mL (500 L / m 2) of the redissolved trastuzumab were loaded onto the device at a flow rate of 1 mL / min (600 LMH), as recommended by the manufacturer. The flowthrough was collected and stored at 4°C prior to analysis.

[0158] 3. 1.9 Pulse injection experiments

[0159] The residence time distribution (RTD) for trastuzumab was determined by pulse injection experiments in all units where the antibody is present as a precipitate (the PEG-1000 tubular reactor and the two TFF stages). The precipitated antibody is retained in the retentate of the HF. To mimic the path followed by the antibody with salt, the permeate lines were closed during the experiments. The system outlet was connected to an Äkta pure 25 (Cytiva) for conductivity recording. First, the system was flushed with deionized water until the conductivity was below 0.3 mS / cm. Pulse injections with a total volume of 10 mL of 1 M NaCl were performed by switching the supply line from deionized water to saline and back. We used the model of series-connected tanks with increasing number (N) of continuously stirred tank reactors (CSTR) to fit our experimental data.The cumulative distribution function (F) for a series of ideally stirred tanks is given by Equation 1: F=1-e. A (-N0) [1 +N0+ (N0) A 2 / 2!+ (N0) A (N-1 ) / (N- 1)1] (1 ) Where 0 denotes the total loading volume [ml] normalized by the system volume [ml].

[0160] 3.1.10 HCPs concentration determination, Bradford assay and ELISA

[0161] For the small-scale screening experiments conducted during process development, the Bradford assay was used for HCP determination, as previously described in the literature (Sissolak et al. 2019). HCP concentration was determined by mass balance on CCCS and the supernatant of samples generated during the experiments after centrifugation of the precipitated or unresolved material at 2000g for 2 min. For continuous processing, HCP determination was performed using a 3rd Generation CHO HCP ELISA Kit (Cignus technologies, Southport, US).

[0162] 3.1.11 hcDNA concentration determination

[0163] The concentration of double-stranded DNA (dsDNA) in the samples generated during the experiments was measured using a Quant-iT Picogreen dsDNA Assay Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions and as previously described in the literature (Dutra et al. 2020).

[0164] 3.1.12 Analytical and preparative Protein A affinity chromatography, analytical size exclusion chromatography (SEC)

[0165] Analytical and preparative protein A affinity chromatography and analytical SEC were performed as described in a previous work (Recanati et al. 2022).

[0166] 3.1.13 Glycosylation analysis

[0167] Antibody samples were analyzed directly on a U3000 nanoRSLC system (Thermo Fisher Scientific). 5 pL of sample was injected onto a C4 trap column (5.0 x 0.3 mm id, Acclaim PepMap; Thermo Fisher Scientific) at a flow rate of 15 pL / min and a mobile phase consisting of H2O + 0.1% trifluoroacetic acid (TFA; Merck, Darmstadt) at 60 °C for 5 min. After antibody capture, the 6-port valve was switched, and the trap was placed in series with the separation column (diphenyl reversed-phase column (15.0 x 0.1 mm id, Halo® Bioclass, 1000 A pore size; Advanced Material Technology, Wilmington, DE). Separation of mAb samples was performed at a flow rate of 1.0 pL / min at 80 °C.A multi-step gradient with solvent A (H2O (ELGA Labwater, Ede, Netherlands) + 0.1% TFA) and solvent B (acetonitrile (Actu-All Chemicals, Oss, Netherlands) + 0.1% TFA) was programmed as follows: 20.0% B for 5 min, 20.0% to 32% in 1.0 min, 32-50% B in 12 min, 50.0-60.0% B in 2 min followed by 90.0% B for 5 min and 20% B for 5 min.

[0168] The LC system was coupled to a qTOF mass spectrometer (Maxis HD) via a nano-ESI source (CaptiveSpray; both from Bruker, Bremen, Germany). Acetonitrile-enriched nitrogen dopant gas was used at a pressure of 0.40 bar (nanoBooster system; Bruker). Drying gas was applied at a flow rate of 3.0 L / min at a temperature of 220 °C. The system was operated in positive ion mode with a capillary voltage of 1200 V. The in-source CID energy was set to 180 eV, the quadrupole ion energy to 5.0 eV, and the collision cell energy to 5.0 eV. A prepulse storage time of 25.0 ps and a transfer time of 200.0 ps were used. The mass range was set from m / z 1000–6000, including a rolling average of three scans. Data analysis was performed using Compass DataAnalysis software (Bruker).Deconvolution of raw mass spectra was achieved using the maximum entropy algorithm, setting a resolution of 5000 and a minimum signal-to-noise ratio of 5.0. 3.2 Results.

[0169] 3.2.1. Development of CaCO and PEGeooo precipitation steps

[0170] CaCl2 precipitation was implemented in the process to reduce the content of soluble hcDNA. Increasing CaCO2 concentrations ranging from 0 to 350 mM at pH 7.0 were added to the CCCS (cell culture supernatant after clarification) (Figure 10A). 100 mM CaCl2 was sufficient to precipitate the majority of the hcDNA present in the clarified cell culture supernatant, and this salt concentration was used for all further experiments.

[0171] The presence of CaCl2 increased the antibody's solubility (Figure 10B). Since Ca2+ is a kosmotropic ion, it promotes salting-in behavior according to the Hofmeister series. Consequently, a higher PEG concentration is required to achieve the same precipitation rate compared to untreated CCCS. The addition of CaCl2 resulted in a twofold and 22-fold reduction of HCPs and hcDNA, respectively. In contrast, extensive coprecipitation of hcDNA, HCPs, and trastuzumab occurred when PEG precipitation was performed on CCCS alone (Figure 10C,D). Coprecipitation complicates the purification of the protein of interest (POI), leading to the simultaneous concentration of impurities and product during tangential flow filtration (TFF). Furthermore, the reduction of hcDNA (in our case before capture) improves the performance of TFF filtration and reduces membrane fouling over time (Mercille et al. 1994).It can be concluded that the addition of CaCL to the CCCS before detection mitigates both of these problems.

[0172] 3.2.2. Depth filter screening and long-term test

[0173] Continuous depth filtration can be achieved by implementing redundancies and operating filters in tandem mode. However, the filter size must match the product mass flow to avoid frequent equipment changeovers, which pose a risk for process errors. We tested double-layer depth filters with different media and pore size ratings from two different suppliers with respect to inlet pressure and turbidity of the filtered stream. All filters reduced the exit turbidity to below 10 NTU. Given this, only the inlet pressure is reported in Figure 11A for simplicity. The Ertelalsop B4E7 depth filter caused the smallest pressure increase during screening.In a long-term experiment, the inlet pressure of this device remained well below the manufacturer's specified pressure threshold (2.4 bar) for approximately 11 hours of operation at a given feed flow of 1.7 ml / min (40 LMH), without increasing turbidity at the outlet (Figure 11B). This filter was selected for use in a continuous process, with an expected change rate of two filters per day.

[0174] 3.2.3. Critical flow assessment for different hollow fiber geometries

[0175] Determining the critical flux is useful for selecting HF modules for specific applications. The precipitate produced under defined conditions and concentrations is recycled on the module while the permeate flow is gradually increased. The critical flux is arbitrarily defined as the permeate flow at which the transmembrane pressure (TMP) increases sharply. For continuous production, HFs are operated well below the critical flux, typically in the range of 1 to 15 LMH. Nevertheless, determining the critical flux can be used as a parameter to compare different modules and select the module that performs best under specific process conditions. A TMP increase corresponding to a maximum of 60 Pa / min is a common threshold. With this fouling rate, an HF can operate for 24 hours before it needs to be replaced (Li and Zidney 2017).

[0176] The inventors investigated membranes with different geometries, such as fiber inner diameter, pore size, and path length. All HFs were tested at a shear rate of 1600 s-1. The critical flux for all tested modules was 109 LMH (Figure 12).

[0177] 3.2.4 Continuous processing, standalone acquisition and integrated perfusion acquisition

[0178] A flow diagram of the entire system is shown in Figure 13. Using the custom-built setup, the inventors ran a continuous process either as standalone acquisition or as integrated perfusion acquisition for up to 8 days. The overall process recovery was 84.5% and 78.4% for the standalone and integrated processes, respectively. A comparison of perfusion productivity and contaminant reduction is shown in Table 1. During standalone acquisition, the trastuzumab titer in the effluent remained constant over the last three days of the run (Figure 14A). Small fluctuations in the product concentration in the effluent are due to the replacement of filters and RF modules (Cytiva, 0.011 m2). During the integrated process, the available modules had a larger membrane area (Asahi Kasei, Microza, 0.02 m2), thus resulting in a larger dead volume.This resulted in higher fluctuations in trastuzumab concentration after replacing the RF modules (Figure 14B).

[0179] Table 1. Comparison of perfusion productivity, recovery of deposition steps, and impurity reduction between deposition alone and integrated perfusion-deposition processes.

[0180] Stand-alone Integrated

[0181] PVP g ■ (L per day) - 1 * 0.4 1.0

[0182] Processed volume (L / 2 2

[0183] Day) purified mAb (g) 4.5 11.9

[0184] HCPs (LRV) 1.1 1 hcDNA (LRV) 1.7 1.9

[0185] Recovery (%) 84.5 78.4

[0186] Nevertheless, the resulting product purity for the two processes was comparable, constant over time, and ranged between 85% and 95% (Figure 15). For both processes, the final concentration of hcDNA and HCPs after capture averaged 30 ppm and 50,000 ppm, respectively. It is noteworthy that during the integrated perfusion capture process, the impurity content in the CCCS varied across all days, whereas the standalone process used a pool and thus maintained a constant CCCS composition throughout the process. Nevertheless, we achieved a similar level of residual impurities for both processes (Figure 16A and B). This underlines the robustness of the precipitation step: the solubility of POI and impurities is determined by the precipitation conditions.For this reason, the ratio of these components is expected to be constant after precipitation (when identical precipitation conditions are applied), as observed in the experiments.

[0187] 3.2.5 Residence time distribution of continuous precipitation and TFF

[0188] The inventors determined the residence time distribution of the system in which the antibody is present in the form of a precipitate (the PEGeooo tubular reactor and the two TFF stages) using pulse spray experiments. Previous work has already demonstrated that the behavior of the precipitated antibody corresponds to that of salt when static mixers are used in tubular reactors (Burgstaller 2019). Therefore, NaCl is used as a tracer for the experiments. Approximately 7 reactor volumes (0) were required to flush all the introduced salt from the system (Figure 17). The average residence time at the given process flow rates and system volume is 15.4 hours. However, this time could be drastically reduced depending on the total system volume, the upstream scale, and the desired conversion rates in the two TFF stages (expressed as a percentage of feed to effluent rates in each stage).As an example, a conversion rate of 95% was applied in the process.

[0189] Depending on the level of soluble contaminants, a conversion rate of 90% or less could be applied. In this case, the average residence time would be halved to 7.8 hours. Furthermore, the reported average residence time for a continuous process of 30 days or more becomes negligible compared to the process duration and the average residence time of the product in the perfusion bioreactor. Regarding further advantageous embodiments of the device according to the invention, reference is made to the general part of the description and the appended claims to avoid repetition.

[0190] Finally, it should be expressly pointed out that the embodiments of the device according to the invention described above serve only to explain the claimed teaching, but do not limit it to the embodiments.

[0191] REFERENCES

[0192] Burgstaller, Continuous recovery of antibodies with non-interrupted mass flow of the product, Vienna, 2019, pp. VII, 38 sheets, 10 unnumbered sheets, 13 sheets, illustrations, diagrams.

[0193] Dutra, D. Komuczki, A. Jungbauer, P. Satzer, Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol, Engineering in Life Sciences 20(7) (2020) 265-274.

[0194] Li, AL Zydney, Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates, Biotechnology Progress 33(6) (2017) 1561 -1567.

[0195] Mercille, M. Johnson, R. Lemieux, B. Massie, Filtration-based perfusion of hybridoma cultures in protein-free medium: Reduction of membrane fouling by medium supplementation with DNase I, Biotechnol Bioeng 43(9) (1994) 833-46.

[0196] Recanati, R. Coca-Whiteford, P. Scheidl, B. Sissolak, A. Jungbauer, Redissolution of recombinant antibodies precipitated by ZnCI2, Process Biochemistry 118 (2022) 145-153.

[0197] Sissolak, C. Zabik, N. Saric, W. Sommeregger, K. Vorauer-Uhl, G. Striedner, Application of the Bradford Assay for Cell Lysis Quantification: Residual Protein Content in Cell Culture Supernatants, Biotechnology Journal 14(7) (2019) 1800714.

Claims

Claims 1. Device for the continuous purification of a biotechnological product, wherein the device has a modular structure with individual interconnected modules, the modules comprising at least one tandem separation module, at least one pump module, and at least one dosing module, characterized in that the device is designed in such a way that an uninterrupted continuous product flow is enabled from the first to the last module.

2. Device according to claim 1, characterized in that • the device is designed to be self-regulating with regard to automatically achieving and maintaining a flow equilibrium of the product flow and / or with regard to pressure distribution within the device; and / or • Functional elements of the modules can be serviced or replaced during operation without interrupting the product flow.

3. Device according to claim 1 or 2, characterized in that the device further comprises at least one sampling module and / or one mixing module.

4. Device according to one of the preceding claims, characterized in that the individual modules are coupled to each other directly or indirectly via a suitable coupling element, preferably sterile.

5. Device according to one of the preceding claims, characterized in that the tandem separation module comprises two identical flow-through separation units, selected from filters and chromatography columns, connected in parallel, wherein the two flow-through separation units can be connected alternately to the product stream, and wherein the tandem separation module preferably includes at least one pressure sensor connected to the The device or module is connected to the control system and is particularly preferably designed to be sterile.

6. Device according to one of the preceding claims, characterized in that the pump module comprises one or more pumps and a volume flow sensor, in particular an ultrasonic volume flow sensor, which is connected to the control of the pump module and ensures constant flow rates.

7. Device according to one of the preceding claims, characterized in that the dosing module is designed to ensure a continuous dosing of additives, wherein the dosing module preferably has a supply container and an intermediate container, between which a filter unit is particularly preferably arranged, which is preferably equipped with a pressure sensor.

8. Device according to one of claims 3 to 7, characterized in that the mixing module is designed to mix several partial streams in a low residence time distribution, wherein the mixing module preferably consists of a hose system with a mixing insert, particularly preferably a disposable article, and preferably has at least one spectrometer for determining the volumetric concentration and / or the mixing efficiency.

9. Device according to one of claims 3 to 8, characterized in that the sampling module is designed to take a sample from a continuous system without interrupting the product flow, wherein the sampling module preferably has a sample receiving container and a buffer container which are arranged to be connected to the product flow simultaneously, so that liquid from the product flow can flow into the sample receiving container and buffer from the buffer container can flow into the main flow at the same rate.

10. Device according to one of claims 3 to 9, characterized in that the device is designed to carry out one or more purification steps and preferably comprises several units built from the modules selected from: • a DNA separation device, particularly preferably comprising a dosing module, a mixing module and a tandem separation module; • a protein separation device, particularly preferably comprising a dosing module, a mixing module and a tandem separation module, • a concentration device, particularly preferably comprising a tandem separation module; • a buffering device, particularly preferably comprising a dosing module, a mixing module and a tandem separation module; • a resolubilization device, particularly preferably comprising a dosing module and a mixing module; and • a virus inactivation device, particularly preferably comprising a dosing module and a mixing module.

11. Device according to one of the preceding claims, characterized in that the device does not contain any intermediate tanks between the modules and / or equipment.

12. A method for the continuous purification of a biotechnological product in a device with a modular design comprising individual interconnected modules, wherein the modules include at least one tandem separation module, at least one pump module, and at least one dosing module, comprising the following steps: - continuous feeding of a product stream containing the biotechnological product and impurities to a first purification unit of the device, wherein the unit comprises at least one tandem separation module, optionally at least one dosing module and optionally a mixing module; - uninterrupted continuous transport of the product stream from one module of the purification system to the next, - Continuous discharge of the product stream with reduced impurities from the facility.

13. Method according to claim 12, characterized in that the biotechnological product is a protein selected from a blood coagulation factor, a peptide hormone, a growth factor, an interferon, an interleukin analogue, or a vaccine, an antibody, preferably antibodies of classes IgG1-4, IgM and IgG, IgE, or derivatives thereof, or a nucleic acid selected from DNA, RNA, such as antisense RNA, or antisense oligonucleotides.

14. Method according to claim 12 or 13, characterized in that the method is implemented without storing the product stream in intermediate tanks and / or is carried out without elution chromatography.

15. Method according to one of claims 12 to 14, characterized in that the device is a device according to one of claims 1 to 11.

16. Method according to one of claims 12 to 15, characterized in that the purification apparatus uses flow-through chromatography or protein precipitation followed by filtration, for example dead-end or tangential flow filtration.

17. A method according to any one of claims 12 to 16, characterized in that the tandem separation module comprises two identical flow-through separation units, selected from filters and chromatography columns, connected in parallel and a pressure sensor, and one of the two flow-through separation units is connected to the product flow and the product flow is switched to the other flow-through separation unit when a pressure threshold is exceeded as a measure of reaching the filling capacity of the flow-through separation unit, wherein preferably the The first flow separation unit is replaced with a new flow separation unit after sterilization of the connecting pieces.

18. Method according to any one of claims 12 to 17, characterized in that additives, such as the precipitant for precipitation, are supplied to the product stream via a dosing module, wherein the dosing module has a supply tank and an intermediate tank, between which a filter unit is arranged which is equipped with a pressure sensor, and wherein the intermediate tank is connected to the product stream and at the same time the supply tank is disconnected from the product stream when the supply tank is empty and / or the pressure at the filter unit exceeds a threshold value.

19. Method according to one of claims 12 to 18, characterized in that a sample is taken from the product stream via a sampling module comprising a sample receiving container and a buffer container without interrupting the product stream, wherein the sample receiving container and the buffer container are connected to the product stream simultaneously, so that liquid flows from the product stream into the sample receiving container and buffer flows from the buffer container into the product stream at the same time.

20. A method according to any one of claims 12 to 19, characterized in that the purification comprises one or more of the following steps in suitable facilities: DNA separation, protein separation, buffering, protein concentration and virus inactivation, wherein preferably the biotechnological product is an antibody and the method comprises each of the aforementioned steps.

21. Method according to any one of claims 12 to 20, characterized in that the steady state of the continuous purification is maintained for a period of at least one week, preferably at least four weeks, particularly preferably at least three months, preferably at least six months.

22. Method according to one of claims 12 to 21, characterized in that the continuous purification is coupled with a continuous protein production.

23. Tandem separation module for use in a process for the continuous purification of a biotechnological product, characterized in that the tandem separation module comprises two identical flow-through separation units, selected from filters and chromatography columns, connected in parallel, wherein the two flow-through separation units can be connected alternately to the product stream, and wherein the tandem separation module has at least one pressure sensor and at least one spectrometer connected to the control of the module, and is particularly preferably designed to be sterilizable.

24. Sampling module for use in a process for the continuous purification of a biotechnological product, characterized in that the sampling module is designed to take a sample from a continuous system without interrupting the product flow, wherein the sampling module preferably comprises a sample receiving container and a buffer container which are arranged to be connected to the product flow simultaneously, so that liquid from the product flow can flow into the sample receiving container and buffer from the buffer container can flow into the product flow simultaneously and to the same extent.

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