DE102022208467A1 - Modular device and method for the continuous production of biotechnological products - Google Patents

Abstract

The present invention relates to a device for the continuous purification of a biotechnological product, the device having a modular structure with individual modules connected to one another, 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 possible from the first to the last module. The invention further relates to a method for the continuous purification of a biotechnological product in a device with a modular structure with individual modules connected to one another, the modules comprising at least one tandem separation module, at least one pump module, and at least one dosing module.

Description

FIELD OF THE INVENTION

The invention relates to a device for the continuous purification of biotechnological products, the device having a modular structure with individual modules connected to one another.

BACKGROUND OF THE INVENTION

A process for the production of biopharmaceutical and biological products usually 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.

Downstream, such a process usually includes the steps of cell separation or the separation of cell fraction components, buffer or media exchange, preferably with concentration, bioburden reduction, preferably with a sterile filter, and capture chromatography. Usually, further steps are carried out to clean the product stream, in particular virus inactivation, neutralization, optionally further BioBurden reduction (with sterile filter). Given the high quality standards in the production of biopharmaceuticals, further steps usually follow: chromatographic intermediate and fine cleaning, bioburden reduction, e.g. with sterile filters, virus filtration as well as buffer exchange and, preferably, concentration.

In order to ensure high purity in the protein purification of monoclonal antibodies (mAb), three consecutive chromatography steps are usually practiced. In the first step of the purification, the mAb should be isolated from the contaminated supernatant in the highest possible yield (capture). The state of the art is purification using affinity chromatography. The antibodies have a region that specifically binds to protein A. Protein A chromatography achieves high yields (>90%) and a large part of the impurities, host cell proteins (HCP) and DNA, are already separated here. The first virus inactivation usually occurs in this step. Elution occurs by reducing the pH value (< 3). Before the eluate is neutralized again, it is kept at a low pH value for a longer period of time in order to carry out virus inactivation.

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

In the field of chromatography, there are efforts to ensure continuous process control. Well-known methods for this are Simulated Moving Bed Chromatography (SMBC) or Periodic Counter Current Chromatography (PCCC). These methods imitate a continuous process control in which several columns are connected to each other in series. The product flow always remains discontinuous.

WO 2012/078677 describes a process and a system for the continuous production of biopharmaceutical products by chromatography and their integration into a production system, in particular into a disposable system. In particular describes the WO 2012/078677 the use of containers (=bags) between units connected in series.

WO2014/137903 describes a solution for the integrated continuous production of a protein substance in a production plant that includes columns for carrying out the production steps, which are connected in series. WO2014/137903 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 in order to minimize production time. WO2014/137903 discloses the use of containers between successive units that can accommodate the product stream for a certain period of time.

EP 3 093 335 A1 describes a modular production system for the continuous production or processing of biopharmaceutical products and a computer-implemented process for process control of the modular system. 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 carry out the step.

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

SUMMARY OF THE INVENTION

The present invention is based, among other things, on the development of tandem separation modules that are suitable for using a device with a truly continuous product flow and can be both 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, the device having a modular structure with individual modules connected to one another, 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 made possible from the first to the last module.

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, the two flow separation units can be alternately connected to the product stream, and wherein the tandem separation module has at least one pressure sensor, which is connected to the control of the module, and is particularly preferably designed to be sterilizable.

Using an optionally included sampling module according to the invention, sampling is achieved without interrupting the mass flow.

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 remove 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 simultaneously connectable to the product stream, so that liquid can flow from the product stream into the sample receiving container and buffer from the buffer container into the product stream at the same time and to the same extent.

To design the device, the individual modules are preferably combined into 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.

• - kontinuierliches Zuführen eines das biotechnologische Produkt und Verunreinigungen enthaltenden Produktstroms zu einer ersten Aufreinigungseinrichtung der Vorrichtung, wobei die Einrichtung mindestens ein Tandem-Abtrennungsmodul, gegebenenfalls mindestens ein Dosierungsmodul und gegebenenfalls ein Durchmischungsmodul umfasst;
• - ununterbrochener kontinuierlicher Transport des Produktstroms von einem Modul der Aufreinigungseinrichtung zum Nächsten,
• - kontinuierliches Ausgeben des Produktstroms mit reduzierten Verunreinigungen aus der Einrichtung.

According to a fourth aspect, the invention relates to a method for the continuous purification of a biotechnological product in a device with 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, comprising the following Steps:

• - continuously feeding a product stream containing the biotechnological product and impurities to a first purification device of the device, the device comprising 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 device to the next,
• - Continuous output of the product stream with reduced impurities from the facility.

CHARACTERS

• 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.
• 2 shows a schematic representation of the dosage module according to the invention.
• 3 shows a schematic representation of the sampling module according to the invention.
• 4 shows a schematic representation of the mixing module according to the invention.
• 5 shows a flowchart of a device according to the invention for the production and purification of antibodies. The individual Different modules are named and modules of the same type are numbered consecutively in the order in which they are used in the process. Same numbers on pumps indicate that they belong to a common pump module.
• 6 shows a schematic representation of a device according to the invention for the production and purification of antibodies, divided into sections AF for clarity. The device includes a cell separation device connected to the bioreactor containing a tandem TFF module ( 6A) ; a DNA separation device containing a dosage module ( 6A) , a mixing module, a tandem dead-end module and a sampling module ( 6 B) ; a protein separation device containing a dosing module, two mixing modules and a tandem TFF module ( 6C) , a concentration and buffering device containing a sampling module, a mixing module and a tandem TFF module ( 6D) ; a virus inactivation device containing a dosage module and a mixing module ( 6 E) Rebuffering device, containing a mixing module ( 6 F) . The numbers on the pumps indicate which pump module they belong to. The inlets and outlets of the individual modules are marked as well as the positions of volume flow and pressure sensors.
• 7 shows a flow chart of a device according to the invention 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. Same numbers on pumps indicate that they belong to a common pump module.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the invention relates to a device for the continuous purification of a biotechnological product, the device having a modular structure with individual modules connected to one another, the modules comprising at least one tandem separation module, at least one pump module, and at least one dosing module, characterized in that that the device is designed in such a way that an uninterrupted, continuous product flow is possible from the first to the last module.

In the context of the invention, the term “product stream” means the fluid from a heterogeneous cell culture fluid mixture which contains the product, as well as the result of all other process steps of the process according to the invention, i.e. the fluid of the process according to the invention flowing between the individual modules, whereby these Product streams can have different concentrations and degrees of purity.

In the context of the invention, a continuous process is understood to mean a process for carrying out at least two successive 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 carried in the production line and is referred to as a “continuous product stream.”

In the context of the invention, the term “closed” means the operation of the method according to the invention and the modular system according to the invention, which are operated in such a way that the product that is produced and/or processed using this method and this modular system is not exposed to the room environment becomes. In the closed process according to the invention and the corresponding closed modular system according to the invention, materials, objects, buffers and the like can be added from the outside, but this addition is carried out in such a way that exposure of the produced and / or processed product to the room environment is avoided .

According to the invention, the device is divided into modules. These modules each have their own process engineering functions. It is possible to replace, add or change the order of individual modules. In the device there are connecting lines between the various modules of the device. The modules can be locked together in a sterile manner. The modules can preferably be connected to one another by welding or using aseptic connectors such as Steam Thru Connectors® or Opta® SFT sterile connectors.

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

According to one embodiment, the device is designed for automatic achievement chens and maintaining a flow equilibrium of the product flow and / or self-regulating with regard to a pressure distribution in the device. According to one embodiment, the device is designed such that functional elements of the modules can be serviced or replaced during ongoing operation without interrupting the product flow.

To automate the device, all pump motors of the device can be adapted to one another and controlled by manual control value specification. For this purpose, one or more pumps are combined in a pump module.

According to one embodiment of the device, the pump module has one or more pumps and volume flow sensors, which are connected to the control of the pump module and ensure constant flow rates. Each pump is preferably connected to an assigned volume flow sensor. The volume flow sensors are particularly preferably ultrasonic volume flow sensors. In particular, these are non-invasive clamp-on ultrasonic sensors, i.e. ultrasonic sensors that are clamped to the outside of the pipe (“clamp-on”), which means that no intervention in the product flow is necessary. According to one embodiment, the pump module has a frame with holders for the individual pumps and the volume flow sensors.

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 adapted to each other by setting simple linear relationships between the pumps in terms of the pump rate (P) or the volumetric flow rate generated $$\text{Z}\text{.B}\text{.:P}1=\text{<figure-callout id="2" label="P" filenames="DE102022208467A1\_0010.png,DE102022208467A1\_0017.png" state="{{state}}">P</figure-callout>}2+\text{<figure-callout id="3" label="P" filenames="DE102022208467A1\_0010.png,DE102022208467A1\_0017.png" state="{{state}}">P</figure-callout>}3\text{or <figure-callout id="1" label="P" filenames="DE102022208467A1\_0010.png,DE102022208467A1\_0017.png" state="{{state}}">P</figure-callout>}1=\text{x*P}$$

If necessary, the pump speed is adjusted to the desired flow rate using a feedback loop. The pumps used can be magnetically coupled pumps or peristaltic pumps. The pumps of a pump module are in particular assigned to different devices of the device. Each pump has an inlet and an outlet that is connected to the assigned modules or connecting lines via aseptic connectors.

The set flow rates or flow speeds 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 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 throughout the process is in the range of 1.0 ml/min to 80 ml/min. According to one embodiment, the flow rate throughout the process is in the range of 2.0 ml/min to 60 ml/min.

According to one embodiment, the modules are controlled individually and independently of one another. In this case there is no communication between the modules in order to avoid error propagation or incorrect control. Alternatively, it is also possible to link the control with an Advanced Process Control System. This allows the process to be optimized and can, for example, enable model predictive control, soft sensing and advanced real-time spectroscopy methods.

Control in the application sense means the measurement of the variable to be influenced (controlled variable) and a continuous comparison with the desired value (setpoint). According to the deviation, a controller calculates a manipulated variable that acts on the controlled variable in such a way that it minimizes the deviation and the controlled variable assumes a desired time behavior. This corresponds to a closed sequence of effects.

In comparison, control refers to the process in a system in which one or more input variables influence the output variables based on the system's own laws. The characteristic feature of the control is the open effect path or a closed effect path, in which the output variables influenced by the input variables do not constantly and do not act on themselves again via the same input variables.

The individual modules can each be coupled to one another directly or indirectly via a suitable coupling element, preferably sterile.

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 connected alternately to the product stream.

The flow separation units are selected from filters and chromatography columns. In particular, it is a tandem tangential flow filter (TFF) or a tandem dead-end filter. 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 drawn off transversely to the direction of flow. The shear forces that occur on the filter surface due to the turbulent flow can be varied depending on the volume flow.

The high speed largely prevents a filter cake (top layer or fouling) from building up on the membrane from the solid particles to be separated. It would increase the filtration resistance and thus the pressure loss across the filter, which reduces the filter performance.

While in the dead-end filter the solids to be separated are obtained as filter cakes, in cross-flow filtration the solids can only be concentrated to such an extent that the suspension can still be pumped. The returned portion of the liquid stream is called retentate.

Alternatively, chromatography columns can be used as flow separation units that contain a chromatography resin with functional groups that do not bind to the desired biotechnological product. Instead, the chromatography columns should have resins that bind to the impurities to be enriched in the product stream.

It is important that the tandem separation modules do not have any chromatography columns that require an elution step, otherwise continuous process control is not possible. In particular, the entire device does not have any separation modules with an elution step.

The tandem separation module can contain at least one pressure sensor. Pressure sensors are preferably connected to the control of the device or module. The load on the filter or chromatography column can be measured using the pressure sensor.

The tandem separation module is designed to ensure sterile parallel connection of the flow separation units (filter or chromatography). The module can be made of stainless steel and can therefore be steamed at 121 °C and 1.1 bar. The module preferably includes a Sterilization-In-Place (SIP) system. This means the module is completely sterilized. As soon as a flow separation unit needs to be replaced, the control can automatically switch to the second unit. The first flow separation unit can be washed and removed. An optionally pre-sterilized filter or an optionally pre-sterilized chromatography column is then inserted as a replacement into the first flow separation unit. A sterilization step can then take place in which the liquid lines that are open during the exchange are sterilized and optionally additionally become the flow separation unit. The unit can then be filled with buffer and switched to the main stream if the module's parallel unit preferably needs to be replaced.

To monitor sterilization, the tandem separation module can have a temperature sensor. The valves are controlled automatically.

Bei Druckanstieg über einen Schwellenwert, z.B. 2 bar Eingangsdruck, kann die Steuerung automatisch auf die parallele Einheit schalten.The tandem dead-end filter module is in 1 B shown. It is the least complex tandem separation module because it does not include a retentate process and the monitoring and control of the system is not very complex. The system can be monitored using pressure sensors, i.e. via the input pressure p _f or the differential pressure $\Delta p=\frac{p_f}{p_R}.$

If the pressure rises above a threshold value, e.g. 2 bar inlet pressure, the control can automatically switch to the parallel unit.

The tandem chromatography module is constructed analogously to the tandem dead-end module. Here too, pressure is monitored, preferably the differential pressure is monitored.

The most complex tandem separation module is the tandem TFF module ( 1A) . According to one embodiment, the tandem TFF module includes holders for the filters, preferably with adjustable clamps, for example with a length in the range of 30 - 65 cm and a diameter in the range of diameter ~ 0.5 - 5 cm. The Tandem TFF module has either a Clean-In-Place (CIP) system or a SIP system. The tandem TFF module has at least three pressure sensors, for the inlet, the retentate and the permeate. With a SIP system, at least one additional pressure sensor is necessary for the steam supply.

The tangential filters are preferably hollow fiber filters or membranes. These can have an area in the range of 100 cm ² - 900 cm ² . The tangential filters have via an inlet and two outlets for permeate and retentate.

• p_F = Druck Zulauf
• p_f = Druck Permeat
• p_R= Druck Retentat

Due to the existing retentate drain, more valves are required and a separation of the two permeate drains may be necessary (due to the sterilization process). The transmembrane pressure (TMP) serves as the control variable. This results from the measured pressures of the feed, the retentate and the permeate. $$TMP=\frac{p_f+{\mathrm{<figure-callout\; id="2"\; label="p\; r"\; filenames="DE102022208467A1\_0010.png,DE102022208467A1\_0017.png"\; state="\{\{state\}\}">p</figure-callout>}}_r}{2}+p_p$$

• p _F = pressure inlet
• p _f = pressure permeate
• p _R = pressure retentate

die Steuerungsgröße sein. Der Schwellenwert für den Transmembran-Drucks (TMP) liegt bei bis zu 2.5 bar und beim der Differentialdruck bei unterhalb von 2 bar.Alternatively, the differential pressure can also be the quotient of the inlet pressure and the retentate pressure $\left({\text{p}}_{\text{R}}\right)\Delta p=\frac{p_f}{p_R}$

be the control variable. The threshold value for the transmembrane pressure (TMP) is up to 2.5 bar and the differential pressure is below 2 bar.

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, which comprises two identical flow separation units, selected from filters and chromatography columns, connected in parallel, the two flow separation units being alternately connectable to the product stream are, and wherein the tandem separation module has at least one pressure sensor, which is connected to the control of the module, and is particularly preferably designed to be sterilizable.

The tandem separation module according to the second aspect is in particular as described in the first aspect.

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

Task of the dosage module ( 2 ) is to ensure 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. In order to maintain the sterility of the system, a dead-end filter capsule can be installed upstream of the intermediate container, which is monitored using a pressure sensor. The supply container can be monitored gravimetrically.

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.

According to one embodiment, the mixing module is designed to mix several partial streams with a low 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 how much time the reaction mass requires on average to flow through a reactor. However, the effective residence times of individual particles can be very different, which results in a residence time distribution.

Depending on the reaction to be carried out, a residence time suitable for the reaction can be defined for the corresponding mixing module 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 from 5 minutes to 60 minutes. For a homogeneous reaction 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 as large as that of a stirred tank with the same volume. In addition, at higher flow speeds, the variance in residence time can be reduced even further. At a flow rate of 7.5 ml/min the variance is only half as large as at around 3 ml/min. The tubular reactor is, in particular, a hose system with a mixing insert. Preferably the mixing module is a disposable item.

In the context of the invention, the term “disposable item” means that the corresponding parts that come into contact with the product, in particular apparatus, Containers, filters and connecting elements, which are suitable for single use with subsequent disposal, these containers can be made of plastic or metal. In the context of the invention, the term also includes reusable objects, for example made of steel, which are only used once in the method according to the invention and are then no longer used in the method. Such used disposable items can then also be referred to as “disposable” or “single-use” items in the method according to the invention (“SU technology”).

According to one embodiment, the mixing module has a spectrometer for determining the volumetric concentration and/or the mixing efficiency.

The mixing module ( 3 ) serves to mix several streams optimally and homogeneously with the lowest possible residence time distribution. It is possible to connect up to two flow cells in order 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. Preferably the mixing module is a disposable item. The mixing module has at least two inputs and at least one output, preferably two outputs.

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

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. At the same time, the path to a buffer container opens which replaces the missing volume flow in the main line in the process direction. After the sample has been taken, the valve turns back to the input position and the product flow flows again in the process direction to the next module. The fill level of the buffer container 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 previously described sampling module.

According to one embodiment, the device is designed to carry out several purification steps. For this purpose, the device preferably has several devices constructed from the modules.

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 buffering 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 contains in particular a dosing module and a mixing module. Another example of such a device is a virus inactivation device. This contains in particular a dosing module and a mixing module.

Due to the modular design, the device can be easily adapted for the purification of any type of protein or nucleic acid. For example, the structure - as shown in Example 2 - can be easily adapted to the purification of proteins from inclusion bodies. In exactly the same way, more protein purification devices can be used as required in order to achieve higher purity if necessary.

The device preferably does not contain any intermediate tanks between the modules and/or devices. Eliminating the need for an intermediate tank is achieved through continuous operation without interruption for the exchange of materials, the elimination of purification modules that require an elution step, and suitable, matching flow rates for the individual process sections.

• - kontinuierliches Zuführen eines das biotechnologische Produkt und Verunreinigungen enthaltenden Produktstroms zur einer ersten Aufreinigungseinrichtung der Vorrichtung, wobei die Einrichtung mindestens ein Tandem-Abtrennungsmodul, gegebenenfalls mindestens ein Dosierungsmodul und gegebenenfalls ein Durchmischungsmodul umfasst;
• - ununterbrochener kontinuierlicher Transport des Produktstroms von einem Modul der Aufreinigungseinrichtung zum Nächsten, und
• - kontinuierliches Ausgeben des Produktstroms mit reduzierten Verunreinigungen aus der Einrichtung.

According to a fourth aspect, the invention relates to a method for the continuous purification of a biotechnological product in a device with 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, comprising the following Steps:

• - continuously feeding a product stream containing the biotechnological product and impurities to a first purification device of the device, the device comprising 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 device to the next, and
• - Continuous output of the product stream with reduced impurities from the facility.

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 clotting factor, a peptide hormone, a growth factor, an interferon, an interleukin analogue, or a vaccine, or an antibody, preferably antibodies of classes IgG1-4, IgM and IgG, IgE, or derivatives thereof .

The protein may also be a protein for food use. The nucleic acid can be selected from DNA, RNA, such as antisense RNA, or antisense oligonucleotides.

According to one embodiment, the process is implemented without storing the product stream in intermediate tanks and/or carried out without elution chromatography. According to an embodiment of the method, the device is a device according to the first aspect.

According to one embodiment, flow-through chromatography or protein precipitation followed by filtration, for example dead-end or tangential flow filtration, is used in the purification device.

und/oder der Druckanstieg bestimmt. Beim Tandem-Dead End-Modul Der Schwellenwert liegt für den TMP im Bereich von 2 bis 2,5 bar. Für den Differentialdruck liegt der Schwellenwert unterhalb von 2 bar.According to one embodiment, the tandem separation module has two identical flow separation units, selected from filters and chromatography columns, connected in parallel and a pressure sensor. One of the two flow separation units is connected to the product stream and when a threshold value for the pressure is exceeded as a measure of reaching the filling capacity of the flow separation unit, the product stream is switched to the other flow separation unit. This switchover can occur automatically or depend on confirmation from the system operator. In a tandem TFF module, the transmembrane pressure (TMP), the differential pressure $\Delta p=\frac{p_f}{p_R}$

and/or the pressure increase is determined. For the tandem dead end module, the threshold value for the TMP is in the range of 2 to 2.5 bar. For the differential pressure, the threshold value is below 2 bar.

When the pressure increases, there is no specific threshold value, but rather an exponential increase, for example, signals that the filling capacity has been reached. As an additional safety function, an alarm signal can usually be triggered below the threshold value.

Then the first flow separation unit can be exchanged for a new flow separation unit while sterilizing the connecting pieces.

According to one embodiment of the method, additives, such as the precipitant for the precipitation, are fed to the product stream via a dosing module. The dosing module has a supply container and an intermediate container, between which a filter unit is arranged, which is equipped with a pressure sensor. The intermediate container is connected to the product stream and at the same time the supply container is separated from the product stream when the supply container is empty and/or the pressure at the filter unit exceeds a threshold value. Pressure at the filter unit above the threshold indicates contamination of the dosing module.

According to one embodiment of the method, a sample is removed 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 connected to the product stream simultaneously, so that at the same time liquid flows from the product stream into the sample receiving container and to the same extent buffer flows from the buffer container into the product stream.

In a further embodiment of the method according to the invention, all units or elements used in modules that come into contact with the product are germ-reduced. In the context of the invention, the term “germ-reduced” means a state of reduced number of germs, i.e. a number of microorganisms men 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 (O ₃ ), hydrogen peroxide treatment (H ₂ O ₂ ) and steam-in- place (SIP) treatment.

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, rebuffering, protein concentration and virus inactivation. In a preferred embodiment, the biotechnological product is an antibody and the method includes each of the steps mentioned. According to one embodiment of the antibody purification, the flow rate in the cell separation device is in the range from 5 ml/min to 10 ml/min, preferably in the range from 6 ml/min to 9 ml/min, particularly preferably in the range from 6.5 ml/min. min to 7.5 ml/min. According to one embodiment, the flow rate in the DNA separation device is in the range from 5 ml/min to 10 ml/min, preferably in the range from 6 ml/min to 9 ml/min, particularly preferably in the range from 7 ml/min to 8 ml/min. 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 buffering 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 of 1.0 ml / min to 2.0 ml / min.

According to one embodiment of the method, the steady state 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 maintaining the individual modules and exchanging materials during ongoing operation, for example through the use of tandem separation modules with flow units.

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

EXAMPLES

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

• • Zellaufschluss bzw. Zellabtrennung
• • DNA Abtrennung bzw. Fällung
• • (selektive) Protein Fällung
• • Konzentrierung
• • Waschen & Umpuffern
• • Resolubilisierung
• • Neutralisation

mAb purification can consist of the following 6 process steps:

• • Cell disruption or cell separation
• • DNA separation or precipitation
• • (selective) protein precipitation
• • Concentration
• • Washing & rebuffering
• • Resolubilization
• • Neutralization

A schematic modular structure of the device for the continuous purification of monoclonal antibodies is shown in 6 shown and as a flowchart in 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.

The dosing module is used to adjust the CaCl ₂ concentration in the product stream to 100 mM from a CaCl ₂ stock solution. It may be necessary to add a minimal concentration of phosphate to the supernatant beforehand. The reaction time for DNA precipitation is a few minutes. An optimal mixture and the necessary residence time are achieved over the length of the tubular reactor module. The precipitate is then separated in the tandem dead-end module using depth filters (23 cm ² to 250 cm ² ). The system is monitored via the pressure (max. 2 - 2.5 bar) and automatically switched to the second filter if the filter fouls. The filter can be replaced, the distance dampened (121°C, 1.1 bar) and reinserted into the system in a sterile manner (functionally closed system).

The protein precipitation takes place in a protein separation device containing a dosing module for the addition of chaotropic substances. The mAb precipitation takes place at a PEG 6000 (polyethylene glycol) concentration of 12 - 15% by volume. This substance is also added from an approx. 50 vol.% stock solution via a dosing module. The reaction time for this precipitation is around 20 minutes and, as in the first step, the optimal residence time and mixing is achieved via a tubular reactor module.

The concentration (5-20 times) takes place in a buffering device with a tandem TFF system. Fluid is continuously removed and new precipitated protein is added. Here, too, the system is monitored via the pressure or transmembrane pressure and, if there is increased fouling of the membrane (pressure increase), it is automatically switched to the second filter. The unused filter can be replaced and the path can be steamed and reinserted into the system in a sterile manner (functionally closed system).

Rebuffering or washing of the precipitate is carried out in a rebuffering device analogous to concentration, except that an additional line with 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 the remaining soluble media and cell culture components. Here too, the system is monitored via pressure and has the redundancy mentioned above.

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

The individual solutions are added via the dosing modules. All flows are monitored with flow sensors and controlled via the associated pumps. The system is continuously in equilibrium. There are no intermediate containers that have a negative impact on the residence time.

Example 2 - Purification and resolubilization of inclusion bodies

• • IB Solubilisierung
• • IB Refolding
• • Proteinlösung umpuffern
• • Proteinlösung aufkonzentrieren

The Inclusion Body (IB) purification and resolubilization includes the following steps:

• • IB solubilization
• • IB refolding
• • Rebuffer protein solution
• • Concentrate protein solution

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

IBs, also called inclusion bodies, arise from a microbial process, namely protein expression in bacteria, particularly Escherichia Coli. During expression, the target protein produced in large quantities is misfolded, aggregation occurs and thus the formation of IBs, in which the target protein is not folded correctly but is present in high purity. After cell rupture, the IBs can be separated from cell debris and remaining medium by centrifugation.

The IB pellet is available as a slurry with a high protein content. If necessary, buffer can be added to the IB Slurry and brought to the desired final protein proportion.

The diluted slurry is introduced into the continuous system via pumps. In the first process step, the IBs are solubilized with a chaotropic or neutral detergent in high molarity, e.g.: 8 M guanidine hydrochloride or 6 M urea. Depending on the product, if di-sulfide bridge bonds have to be formed, this process must already take place under reducing operations. This usually happens with the addition of dithiothreitol (DTT), e.g.: 100 mM.

After solubilization, the protein refolds. By rebuffering, for example with a 100mM 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.

In the last step, the (usually) diluted protein solution coming from the process is concentrated.

The tandem filter modules used correspond to those from Example 1. The same control strategies are also used. However, due to the size of the IBs, (hollow fiber) filter membranes with a different cut-off 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 cut-off of 0.2 µm is used. From the solubilization step onwards, membranes with cut-offs 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 is present as IB can be continuously purified with this process setup. Examples of industry-relevant products are insulin, FAb fragments (fragments of monoclonal antibodies) and casein.

With regard to further advantageous embodiments of the device according to the invention, in order to avoid repetitions in general Reference is made to part of the description and to the attached claims.

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

QUOTES INCLUDED IN THE DESCRIPTION

This list of documents listed by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2012078677 [0007]
• WO 2014137903 [0008]
• EP 3093335 A1 [0009]

Claims (24)

Device for the continuous purification of a biotechnological product, the device having a modular structure with individual modules connected to one another, 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 that an uninterrupted, continuous product flow is possible from the first to the last module. 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 stream and/or with regard to pressure distribution in the device; and/or • Functional elements of the modules can be maintained or replaced during ongoing operation without interrupting the product flow. Device according to Claim 1 or 2 , characterized in that the device further has at least one sampling module and/or a mixing module. Device according to one of the preceding claims, characterized in that the individual modules are each coupled to one another directly or indirectly via a suitable coupling element, preferably sterile. Device according to one of the preceding claims, 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 can be connected alternately to the product stream, and wherein the tandem separation module preferably at least a pressure sensor which is connected to the control of the device or the module and is particularly preferably designed to be sterilizable. Device according to one of the preceding claims, characterized in that the pump module has 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. Device according to one of the preceding claims, characterized in that the dosing module is designed to ensure a continuous dosing of additives, the dosing module preferably having a supply container and an intermediate container, between which a filter unit is particularly preferably arranged, which is preferably equipped with a pressure sensor Is provided. Device according to one of the Claims 3 until 7 , characterized in that the mixing module is designed to mix several partial streams with a low residence time distribution, the mixing module preferably consisting of a hose system with a mixing insert, particularly preferably a disposable item, and preferably at least one spectrometer for determining the volumetric concentration and / or the Has mixing efficiency. Device according to one of the Claims 3 until 8th , characterized in that the sampling module is designed to remove a sample from a continuous system without interrupting the product flow, the sampling module preferably having a sample receiving container and a buffer container, which are arranged to be simultaneously connectable to the product flow, so that at the same time to the same extent liquid can flow from the product stream into the sample receiving container and buffer can flow from the buffer container into the main stream. Device according to one of the Claims 3 until 9 , characterized in that the device is designed to carry out one or more purification steps and preferably has several devices constructed from the modules, selected from: • a DNA separation device, particularly preferably containing a dosing module, a mixing module and a tandem separation module; • a protein separation device, particularly preferably containing a dosing module, a mixing module and a tandem separation module, • a concentration device, particularly preferably containing a tandem separation module; • a buffering device, particularly preferably containing a dosing module, a mixing module and a tandem separation module; • a resolubilization device, particularly preferably containing a dosing module and a mixing module; and • a virus inactivation device, particularly preferably containing a dosing module and a mixing module. Device according to one of the preceding claims, characterized in that the Device does not contain any intermediate tanks between the modules and / or devices. Method for the continuous purification of a biotechnological product in a device with 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, comprising the following steps: - continuously feeding a product stream containing the biotechnological product and impurities to a first purification device of the device, the device comprising 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 device to the next, - Continuous output of the product stream with reduced impurities from the facility. Procedure according to Claim 12 , characterized in that the biotechnological product is a protein selected from a blood clotting 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. Procedure according to Claim 12 or 13 , characterized in that the process is implemented without storing the product stream in intermediate tanks and/or is carried out without elution chromatography. Procedure according to one of the Claims 12 until 14 , characterized in that the device is a device according to one of the Claims 1 until 11 is. Procedure according to one of the Claims 12 until 15 , characterized in that flow-through chromatography or protein precipitation followed by filtration, for example dead-end or tangential flow filtration, is used in the purification device. Procedure according to one of the Claims 12 until 16 , characterized in that the tandem separation module comprises two identical flow separation units, selected from filters and chromatography columns, in parallel connection and a pressure sensor, and one of the two flow separation units is connected to the product stream and the product stream when a threshold value for the pressure is exceeded as Measure for reaching the filling capacity of the flow separation unit, to which the other flow separation unit is switched, the first flow separation unit preferably being exchanged for a new flow separation unit while sterilizing the connecting pieces. Procedure according to one of the Claims 12 until 17 , characterized in that additives, such as the precipitant for the precipitation, are supplied to the product stream via a dosing module, the dosing module having a supply container and an intermediate container, between which a filter unit is arranged, which is equipped with a pressure sensor, and wherein the intermediate container connected to the product stream and at the same time the supply container is separated from the product stream when the supply container is empty and / or the pressure at the filter unit exceeds a threshold value. Procedure according to one of the Claims 12 until 18 , characterized in that a sample is removed from the product stream via a sampling module, comprising a sample receptacle and a buffer container, without interrupting the product stream, the sample receptacle and the buffer container being connected to the product stream simultaneously, so that liquid is simultaneously released from the product stream Product stream flows into the sample receiving container and to the same extent buffer flows from the buffer container into the product stream. Procedure according to one of the Claims 12 until 19 , characterized in that the purification comprises one or more of the following steps in suitable facilities: DNA separation, protein separation, re-buffering, protein concentration and virus inactivation, the biotechnological product preferably being an antibody and the method comprising each of the steps mentioned. Procedure according to one of the Claims 12 until 20 , characterized in that the steady state 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. Procedure according to one of the Claims 12 until 21 , characterized in that the continuous purification is coupled with continuous protein production. 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, the two flow separation units being alternately connectable to the product stream, and wherein the tandem separation module has at least one pressure sensor and at least one spectrometer, which are connected to the control of the module, and is particularly preferably designed to be sterilizable. Sampling module for use in a method for the continuous purification of a biotechnological product, characterized in that the sampling module is designed to remove a sample from a continuous system without interrupting the product flow, the sampling module preferably having a sample receiving container and a buffer container, which are arranged to be simultaneously connectable to the product stream, so that liquid can flow from the product stream into the sample receiving container and buffer from the buffer container into the product stream at the same time and to the same extent.

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