WO2023247798A1

WO2023247798A1 - Modular device and method for continuously producing biotechnological products

Info

Publication number: WO2023247798A1
Application number: PCT/EP2023/067288
Authority: WO
Inventors: Bernhard SISSOLAK; Alois Jungbauer; Sebastián Avelino GÓMEZ SÁNCHEZ; Magdalena PAPPENREITER; Gabriele RECANATI; Laura Sandy BARBU
Original assignee: Bilfinger Life Science Gmbh
Priority date: 2022-06-24
Filing date: 2023-06-26
Publication date: 2023-12-28

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

MODULAR DEVICE AND METHOD FOR THE CONTINUOUS PRODUCTION OF BIOTECHNOLOGICAL PRODUCTS

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> 1 m). 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 method 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, WO 2012/078677 describes the use of containers (=bags) between units connected in series.

WO 2014/137903 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 that are connected in series. WHERE 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 in order to minimize production time. WO 2014/137903 discloses the use of containers between successive units that can accommodate the product flow for a certain time.

EP 3 093 335 A1 describes a modular production system for the continuous production or preparation of biopharmaceutical products and a computer-implemented method for process control of the modular system. According to EP 3 093 335 A1, a module or a 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 containers, 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 used to carry out the individual steps of the purification process are suitable, for example a DNA separation device, a protein separation device, a concentration device, or a rebuffering device, a resolubilization device.

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.

Fig. 2 shows a schematic representation of the invention

dosage module.

Fig. 3 shows a schematic representation of the invention

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

Fig. 5 shows a flow chart of a device 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. Same numbers on pumps indicate that they belong to a common pump module.

Fig. 6 shows a schematic representation of a device according to the invention for the production and purification of antibodies, divided into sections A-F for clarity. The device includes, 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

Rebuffering 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). Rebuffering device, containing a mixing module (Fig. 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.

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

Figure 9 shows the transmembrane and axial pressure of two interconnected TFF modules over the process run. Increase in pressure indicates fouling of the membrane. Switching to the second filter results in a pressure drop. There were six switches in the first stage and two in the second stage.

Fig. 10 shows the process development for CaCl2 and PEGeooo precipitation steps. A) Concentration of soluble hcDNA with increasing CaCl2 concentrations (0-350 mM). B, C, D) Trastuzumab precipitation yield, concentration of soluble hcDNA, concentration of soluble HCPs with (white circles, dashed line) or without (black circles, solid line) CaCO 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.

Figure 11 shows screening and testing of depth filters to remove precipitated hcDNA after adding 100 mM CaCl final concentration to the cell culture supernatant. A) Screening of depth filters at a volumetric load of 250 L/m2. B) Long-term test for the Ertelalsp B4E7 depth filter for use in a continuous process. Both the screening and the long-term test were carried out at a flow rate of 1.7 mL/min (40 LMH).

Fig. 12 shows experiments on the critical flux of hollow fiber modules in recycling mode at a shear rate of 1600 s-1 and a concentration of precipitated monoclonal antibodies of 5 g/L in 18% PEGeooo, 100 mM MOPS buffer, pH 7. 0 were carried out. Pore size and inner diameter (ID) for each module are indicated in the figure legend, as is the maximum critical flow (LMH). Modules #1, #2 and #3 had a path length of 30 cm. Only module #4 had a path length of 20 cm.

Figure 13 shows a flowchart of the integrated perfusion capture setup. The clarified cell culture supernatant from the perfusion bioreactor cell retention device (TFF) is subjected to two successive precipitation stages (CaCO or PEGeooo), which are separated from each other by a depth filtration stage. The precipitated antibody is concentrated in the first stage of TFF and passed to the second stage of TFF where it is washed by adding fresh buffer, reconcentrated and collected as a precipitate.

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

Figure 15 shows the determination of aggregates and monomers (%) based on 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 independent recording over seven days of continuous process. B) Aggregates and monomers (%) for integrated perfusion deposition over an eight-day process duration. C) Aggregates and monomers (%) for cell culture Supernatant samples from the perfusion process over eight days of process integration for integrated perfusion capture.

Figure 16 shows the concentration (ppm) of hcDNA (A) and HCPs (B) for the cell culture supernatant used as starting material for the stand-alone capture (white triangle, dashed line), for the stand-alone -Capture obtained redissolved trastuzumab (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). ).

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 shown by Application of the tank-in-series model for a series of CSTRs of 1, 5 and 10 CSTRs was obtained, respectively.

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, 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 called 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 can be operated in such a way that the product that is produced and/or processed using this method and this modular system does not leak into the room environment is exposed. 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 welded or through aseptic connectors such as Steam Thru Connectors®, or Opta® SFT sterile connector.

This uninterrupted continuous flow of product is achieved through an appropriate 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 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. 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 that 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 pumps in terms of pumping rate (P) or volumetric flow rate produced.

E.g.: P1 = P2+P3 or P1 = 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, which are 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 is throughout

Process in the range from 1.0 ml / min to 80 ml / min. According to one embodiment, the flow rate is throughout

Process range from 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, model predictive control, soft sensor technology and advanced real-time spectroscopy methods are possible.

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 into the first as a replacement

Flow separation unit used. 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.

The tandem dead-end filter module is shown in Figure 1B. 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 pf or the differential pressure Ap = pf / PR. 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 (Fig. 1 A). 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 ~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 one inlet and two outlets for permeate and retentate.

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 = (pt + p _r ) / 2 +p _P

PF = pressure inlet pp = pressure permeate PR= pressure retentate

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 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.

The task of the dosing module (Fig. 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 needs on average to flow through a reactor. Included 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, are suitable for single use with subsequent disposal, whereby 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 (Fig. 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. This eliminates the need for an intermediate tank Permanent operation without interruption for the exchange of materials, the elimination of purification modules that require an elution step, and suitable, matching flow velocities of the individual process sections are achieved.

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

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.

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 Ap = pt / PR and/or the pressure rise are 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.

The tandem operation allows continuous switching back and forth between the units (depth filters, membranes, etc.). The pressure monitoring prevents the critical pressure from being exceeded and thus enables continuous operation (see also Figure 9). At increased pressure, the mass flow is directed to the second stage. This creates a pressure drop. The process is not stopped and continues continuously. The state of equilibrium is maintained.

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.

The main flow can be regulated using a cylinder valve designed by yourself, taking into account the 3D rule. To seal the sampling module, for example, an O-ring (D = 2 mm) and a pin are integrated into the cylinder valve and polytetrafluoroethylene (PTFE) seals are inserted between the tri-clamps.

The sensors are used to 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 system process, an alarm would be triggered if the pressure was exceeded. The present sampling loop is suitable for taking a sample of 2 ml. The advantage of this construction is that Adjust the sample amount by replacing the sampling loop with a longer sampling loop.

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 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.

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 Zmin up 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. 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 preferred in the range from 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

mAb purification can consist of the following 6 process steps:

• Cell disruption or cell separation

• DNA separation or precipitation

• (selective) protein precipitation

• Concentration

• Wash & Buffer

• Resolubilization

• Neutralization

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

The dosing module is used to adjust the CaCl concentration in the product stream to 100 mM from a CaCl2 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 path is 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 PEGeooo (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

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 FIG.

IBs, also called inclusion bodies, arise from a microbial process, namely protein expression in bacteria, especially 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 pm 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.

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

3.1 Methods

3.1.1 Cell culture

A recombinant IgG1 (trade name Trastuzumab, PI 8.4-8.6) was used in this study. CHO cells were cultured in perfusion mode in a Labfors 5 benchtop bioreactor (Infors Ht, Switzerland). Cell proliferation was carried out in shake flask cultures (Corning, New York, USA) with a working volume of up to 500 mL. The bioreactors were seeded with a cell density of 3-5 x 10 ⁶ cells/mL. Perfusion was initiated on the same day with 0.5 WD by continuously supplying fresh perfusion medium and removing harvest from the permeate. A perfusion rate of 0.5 WD was maintained until a cell density of 10 x 10 ⁶ cells/mL was reached and was gradually increased with cell density up to 1 WD until the live cell concentration (VCC) set point of 60 ± 10 x 10 ⁶ cells/mL was reached. Cell bleed was applied via an automated bleed feedback loop with VCC prediction from an online capacitance probe (Incyte, Hamilton, Switzerland). The working volume was maintained at 2 L by maintaining constant inflow and outflow rates. Feedback loops of bioreactor weight, feed, and bleed were used to adjust 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 of the process temperature, dissolved oxygen concentration and culture pH Values 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 capture 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 capture, 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.

3.1.2 hcDNA precipitation by CaCF

To determine the best conditions for efficient hcDNA precipitation, increasing concentrations of CaCl2 from 0 to 350 mM from a 1 M stock solution of CaCl in 100 mM MOPS, pH 7.0, were added to the CCCS. The screening was carried out in 1.5 mL centrifuge tubes with a total working volume of 1 mL. The samples were gently shaken for 5 minutes on the 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 min and the supernatant was collected in fresh centrifuge tubes for subsequent analysis.

3.7.3 Deep filtration of the precipitated impurities

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

3. 1.4 Trastuzumab precipitation by PEGeooo

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 carried out in

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

3. 1.5 Critical Flow Experiments

Critical flow experiments were conducted on different HFs according to Li and Zidney 2017. The precipitation product was pumped with a peristaltic pump (313 DV Watson Marlow, Guntramsdorf, Austria) from a 100 mL reservoir at a concentration of 5 g/L to the inlet of the HF, 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 in a range from 11 LMH to 109 LMH. Transmembrane pressure (TMP) was monitored by installing laboratory-scale pressure sensors (PendoTech, New Jersey, USA) on inlet, retentate and permeate lines of the HF. The pressure sensors were connected to a PressureMAT® (PendoTech, New Jersey, USA) and Qubicon® online software was used for monitoring and recording.

3. 1.6 Resolubization of trastuzumab at low pH

Resolubization of captured trastuzumab was performed batchwise with a 50 mM phosphate buffer pH 2.5 at a sample:buffer volumetric dilution ratio of 1:5. The samples were incubated for 60 minutes at low pH (<3.5) on the end-to-end shaker (Stuart Rotator SB3; Cole-Parmer, Vernon Hills, IL) and by adding 200 mM phosphate buffer pH 8, 0 neutralized with a volumetric dilution ratio sample:buffer 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.

3.1.7 Continuous 2-stage precipitation and 2-stage TFF

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 precipitation of trastuzumab or directly to the first precipitation stage in a poolless integrated perfusion capture. Stock solutions of CaCL and PEGeooo were prepared according to the previous experiments. All solutions were sterile filtered using 0.2 pm filters Sartopore® 2 XLG capsule (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 by inserting static mixers (HT-40-6.30-24-AC; material acetal; Stamixco AG, Wollerau, Switzerland) into standard flexible laboratory tubing (Tygon® R-3603, 4.8 mm inner diameter; Saint- Gobain, Courbevoie, France) were inserted, which were arranged in a spiral and stacked vertically. The length of the tubular reactors was determined according to the residence time required for efficient precipitation (5 minutes for CaCL precipitation, 15 minutes for PEGeooo precipitation).

3. 1.8 Flow Anion Exchange Membrane Chromatography

The precipitate harvested during the integrated perfusion capture process was dissolved at low pH as reported in Section 2.6. We used a DoE approach to optimize the process. The software used for the design of the experiments and the 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 diluting 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 redissolved trastuzumab was loaded onto the device at a flow rate of 1 mL/min (600 LMH) as suggested by the manufacturer. The flowthrough was collected and stored at 4°C prior to analysis.

3. 1.9 Pulse injection experiments

The residence time distribution (RTD) for trastuzumab was determined by pulse injection experiments in all units where the antibody is in the form of a precipitate (the PEGeooo tubular reactor and the two TFF stages). The precipitated antibody is retained in the retentate of the HF. To mimic the path that the antibody follows with salt, the permeate lines were closed during the experiments. The output of the system 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 feed line from deionized water to saline and back. We have the model in series switched tanks with increasing numbers (N) of continuously stirred tank reactors (CSTR) were used 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].

3.1.10 HCPs concentration determination, Bradford assay and ELISA

For the small-scale screening experiments performed during process development, the Bradford assay was used for HCPs determination, as previously described in the literature (Sissolak et al. 2019). The HC Ps 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 2000 g for 2 min. For continuous processing, HCPs determination was carried out using a 3rd generation CHO HCP ELISA kit (Cignus technologies, Southport, US).

3.1.11 hcDNA concentration determination

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).

3.1.12 Analytical and preparative protein A affinity chromatography, analytical size exclusion chromatography (SEC)

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

3.1.13 Glycosylation analysis

Antibody samples were analyzed directly on a U3000 nanoRSLC system (Thermo Fisher Scientific). 5 pL of the sample was added to a C4 trap column (5.0 x 0.3mm id, Acclaim PepMap; Thermo Fisher Scientific), with a flow rate of 15 pL/min and a mobile phase consisting of H2O + 0.1% trifluoroacetic acid (TFA; Merck, Darmstadt), at a temperature of 60 °C for 5 min. After capture Antibody, the 6-port valve was switched and the trap was placed in series with the separation column (diphenyl reverse phase column (15.0 x 0.1 mm ID, Halo® Bioclass, 1000 A pore size; Advanced Material Technology, Wilmington, DE). The separation of the mAb samples was carried out with a flow rate of 1.0 pL/min at a temperature of 80 ° C. A multi-stage 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.

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 lone energy to 5.0, 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 by the maximum entropy algorithm by setting a resolution of 5000 and a minimum signal-to-noise ratio of 5.0. 3.2 Results

3.2.1. Development of CaCO and PEGeooo precipitation steps

CaCl2 precipitation was implemented in the process to reduce the soluble hcDNA content. Increasing CaCO 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 most of the hcDNA present in the clarified cell culture supernatant, and this salt concentration was used for all further experiments.

The presence of CaCl2 increased the solubility of the antibody (Figure 10B). Since Ca2+ is a cosmotropic ion, it favors salting-in behavior according to the Hofmeister series. As a result, a higher PEG concentration is necessary to achieve the same precipitation rate compared to the untreated CCCS. The addition of CaCL resulted in a reduction of HCPs and hcDNA by 2-fold and 22-fold, respectively. In contrast, there was extensive co-precipitation of hcDNA, HCPs and trastuzumab when PEGeooo precipitation was performed on CCCS alone (Figure 10 C,D). Co-precipitation makes purification of the protein of interest (POI) difficult, resulting in the simultaneous concentration of impurities and product during tangential flow filtration (TFF). Furthermore, the reduction of hcDNA (in our case before acquisition) 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 capture reduces both problems mentioned.

3.2.2. Depth filter screening and long-term test

Continuous depth filtration can be achieved by implementing redundancies and operating filters in tandem mode. However, the size of the filters must match the product mass flow to avoid frequent equipment changes, which pose a risk of process errors. We examined dual layer depth filters with different media and pore size ratings from two different vendors for inlet pressure and filtered stream turbidity. All filters reduced the turbidity at the output to less than 10 NTU. In view of this, only the inlet pressure is reported in Figure 11A for simplicity. The Ertelalsop B4E7 depth filter caused the lowest 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 an increase in turbidity at the outlet (Figure 11 B). . This filter was selected for use in a continuous process, with an expected change rate of two filters per day.

3.2.3. Critical flow evaluation for different hollow fiber geometries

Critical flow determination is useful for selecting RF modules for specific applications. The precipitate material produced under defined conditions and concentrations is recycled on the module while the permeate flow is gradually increased. The critical flow 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 flow and typically in the range of 1 to 15 LMH. Nevertheless, the determination of the critical flow can be used as a parameter to compare different modules and to select the module that performs best in specific process conditions. A TMP increase corresponding to a maximum of 60 Pa/min is a common threshold. At this contamination rate, an HF can operate for 24 hours before needing to be replaced (Li and Zidney 2017).

The inventors examined 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 flow for all modules tested was 109 LMH (Figure 12).

3.2.4 Continuous processing, standalone acquisition and integrated perfusion acquisition

A flowchart of the entire system is shown in Figure 13. With the customized facility, the inventors have a continuous process performed either as a stand-alone recording or as an integrated perfusion recording for up to 8 days. The total 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. When recorded independently, the trastuzumab titer in effluent remained constant over the last three days of the run (Figure 14A). Small fluctuations in product concentration in the drain are due to the replacement of filters and HF modules (Cytiva, 0.011 m2). During the integrated process, the available modules had a larger membrane area (Asahi Kasei, Microza, 0.02 m2), therefore a larger dead volume. This resulted in higher fluctuations in trastuzumab concentration after replacing the RF modules (Figure 14B).

Table 1. Comparison of perfusion productivity, capture step recovery, and contaminant reduction for standalone capture and integrated perfusion capture processes.

Stand-alone Integrated

PVP g ■ (L per day) - ¹ * 0.4 1.0

Processed volume (L / 2 2

day) purified mAb (g) 4.5 11.9

HCPs (LRV) 1.1 1 hcDNA (LRV) 1.7 1.9

Recovery (%) 84.5 78.4

Nevertheless, the resulting product purity for the two processes was comparable, constant over time and 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 detection process, the content of impurities in the CCCS varied on all days, while in the stand-alone process a pool was used and thus there was a constant composition of the CCCS throughout the entire process. Nevertheless, we achieved similar levels 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 found in the experiments.

3.2.5 Residence time distribution of continuous precipitation and TFF

The inventors determined the residence time distribution of the system in which the antibody is in the form of precipitation (the PEGeooo tube reactor and the two stages of the TFF) through pulse spray experiments. Previous work has already proven that the behavior of the precipitated antibody corresponds to the behavior 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 necessary to flush all of 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 volume of the system, the upstream scale, and the desired conversion rates in the two TFF stages (expressed as a percentage of feed to outflow rates in each stage). As an example, a conversion rate of 95% was used in the process.

Depending on the level of soluble impurities, 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, for a continuous process of 30 days or more, the reported average residence time becomes negligible compared to the process duration and the average residence time of the product in the perfusion bioreactor. With regard to further advantageous embodiments of the device according to the invention, in order to avoid repetition, reference is made to the general 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.

CREDENTIALS

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

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.

Li, A.L. 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.

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 .

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

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

Expectations

1. 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 this way is 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 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.

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

4. Device according to one of the preceding claims, characterized in that the individual modules are coupled to one another 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 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 preferably at least one pressure sensor that is connected to the Control of the device or module is connected, and is particularly preferably designed to be sterilizable.

6. 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.

7. 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 with is 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 small 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 mixing efficiency.

9. Device according to one of claims 3 to 8, 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 with 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 from the buffer container into the main stream to the same extent.

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 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.

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 devices.

12. 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,

13. The method 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 the IgG1 class -4, IgM and IgG, IgE, or derivatives thereof, or a nucleic acid selected from DNA, RNA, such as antisense RNA, or antisense oligonucleotides.

14. The 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. The method according to any one of claims 12 to 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.

17. The method according to any one of claims 12 to 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 flow is switched to the other flow separation unit when a threshold value for the pressure is exceeded as a measure of reaching the filling capacity of the flow separation unit, preferably the first flow separation unit is replaced with a new flow separation unit with sterilization of the connecting pieces.

18. The method according to any one of claims 12 to 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 is equipped with a pressure sensor, and wherein 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.

19. The method according to any one of claims 12 to 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 flow, the sample receptacle and the buffer container being connected to the product stream be connected 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.

20. The 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, rebuffering, protein concentration and virus inactivation, the biotechnological product preferably being an antibody and the procedure includes each of the steps mentioned.

21. The method according to any one of claims 12 to 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.

22. The method according to any one of claims 12 to 21, characterized in that the continuous purification is coupled with 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 separation units, selected from filters and chromatography columns, in parallel connection, 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 and at least one spectrometer, which are connected to the control of the module, and is particularly preferably designed to be sterilizable.

24. 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.