Patent Publication Number: US-2020291344-A1

Title: Device and method for examining a medium

Description:
The invention relates to a device and a method for examining a medium inside a bioreactor, in particular a disposable bioreactor. 
     The trend in the biopharmaceutical industry is increasingly towards so-called disposable bioreactors, such as bags made of plastic film, which are also called bags. The expectation of end users of these disposable bioreactors is to have the same systems, measurement methods and parameters available as in classic stainless steel bioreactors. In particular, the measuring methods used should meet the same requirements as for stainless steel bioreactors in terms of their accuracy, reliability and the measuring range to be examined. To this end, various sensors, such as optical pH sensors and optical sensors for dissolved oxygen, are already integrated in the disposable bioreactors. This happens inter alia by welding special ports and special holding devices into the reactor wall of the disposable bioreactor. 
     A measurement of nutrients such as glucose or glutamine or metabolites such as lactate or glutamate is currently only possible by manual sampling and subsequent laboratory analysis or a stainless steel immersion probe. The immersion probes are designed in such a way that they can extract a continuous, representative amount of medium from the reactor through a membrane, by dialysis or filtration, and feed it to an analysis system. 
     A measurement of a target protein, e.g., a monoclonal antibody, is only possible by sampling, additional purification and subsequent laboratory analysis (HPLC or ELISA techniques). 
     The customary method for sterilizing disposable bioreactors is irradiation with up to 50 kGy gamma radiation. In order to keep disposable bioreactors sterile during sampling, it is necessary that an insertion of an immersion probe for continuous cell-free sampling be only possible via a special port, with particular attention to hygiene conditions. However, using such a probe can result in unwanted contamination of the cell culture process or damage to the disposable bioreactor. 
     Disposable bioreactors currently used are basically available in three main types. Stirred systems with impeller and sparger for gassing and agitation, shaken systems and systems realizing ventilation and mixing via wave movement, which are also referred to as RM for “rocking motion”, In particular in current RM systems, it is not readily possible to insert stainless steel immersion probes due to the design of the disposable bioreactor and the movement which prevents permanent immersion of the immersion probes. 
     It is the object of the invention to improve the examination and/or purification of a medium inside a bioreactor, 
     This object is solved by the subject matters of the independent claims. Preferred embodiments are the subject of the dependent claims. 
     One aspect relates to a device for examining a medium inside a bioreactor, in particular a disposable bioreactor, with a sampling module for taking a sample of the medium. The sampling module has an extraction region that can be arranged in touch-contact with the medium inside the bioreactor. At least two different membranes for taking a sample of the medium are arranged at the extraction region of the sampling module. 
     The device is designed and provided to examine online the medium arranged inside a bioreactor, i.e., essentially without disturbing and/or interrupting the biological process inside the bioreactor. Even if the device is designed to examine the contents of a disposable bioreactor, the device can also be designed to examine the contents of a stainless steel bioreactor and/or a pallet tank. 
     The sampling module can be designed as an immersion probe and/or can have an immersion probe, in particular an immersion probe made of stainless steel. The sampling module can also have a welding port and/or be designed as a welding port, in which the membrane(s) is/are arranged. Here, the welding port can be flat, e.g., have a bioreactor surface facing the interior of the bioreactor and/or an outer surface facing away from the interior of the bioreactor. Supply lines to the welding port can be arranged on a side of the sampling module facing away from the interior of the bioreactor, e.g., on the above-mentioned outer surface of the welding port. The sampling module can also have a hose and/or can be substantially hose-shaped. Here, at least one of the membranes can be designed as part of the hose wall. Furthermore, the sampling module can also have a tube and/or be essentially tubular. 
     The sampling module can also be used for the targeted purification of the medium of the bioreactor. 
     The extraction region of the sampling module can be designed and provided to be inserted into the interior of the bioreactor in an operating position. In the operating position of the device, the extraction region is arranged in touch-contact with the (e.g., biological) medium inside the bioreactor. 
     The extraction region can e.g., be designed as a probe part, which e.g., contains a probe tip. The extraction region can have a probe tip. 
     In the operating position, the extraction region can be guided into the interior of the bioreactor e.g., from the outside through a port in the reactor wall of the bioreactor. 
     The sampling module can have an external module region arranged outside the bioreactor in the operating position. The extraction region can be connected to the external module region via at least one transport line through the interior of the sampling module, in which a sample of the medium can be transported out of the bioreactor. 
     The sampling module can be designed to be secured to a holder on and/or relative to the bioreactor in such a way that the extraction region projects through the reactor wall into the interior of the bioreactor, while the external module region of the sampling module is arranged outside the bioreactor. 
     The extraction region can have a hollow inner region, into which the sample(s) of the medium can be introduced. The hollow inner region can be designed as part of the transport line. 
     The sampling module serves to take a sample of the medium from the interior of the bioreactor. Here, dialytic sampling can take place, wherein a sample is taken without reducing the total volume. The medium is usually a mixture of different extraction region such as cells, proteins, antibodies and/or smaller molecules such as e.g., glucose. Taking a sample here means that a sample is taken from at least one of these components of the medium, not necessarily a fraction of all different components of the medium. Here, tubular cleaning modules can be used, e.g., Filer devices. 
     The sample of the medium taken can be examined after sampling, e.g., after it has first been transported to the external module region of the sampling module and from there to an analysis module that does the actual analysis. The sampling module can e.g be connected and/or coupled to an external analysis system, e.g., to a mass spectrometer, a gas chromatograph, a liquid chromatography system such as an HPLC/UPLC system (which stands for High Performance Liquid Chromatography or Ultra Performance Liquid Chromatography) and/or an enzymatic analysis system such as Trace. 
     At least two different membranes for taking a sample of the medium are arranged at the extraction region of the sampling module. The two membranes can e.g., differ in which components of the medium can be taken as samples. The membranes can differ in particular in their cutoff pore size and/or can be designed to be differently permeable and/or to be of different strengths. The sampling module can in particular have exactly two different membranes for sampling. Alternatively, the sampling module can also have more than two different membranes. 
     As explained at the beginning, there have so far only been immersion probes for determining the concentration of nutrients. These have a specific membrane that is permeable to nutrients. 
     As to the examination of the medium, the invention is not limited to this one component of the medium, as in the case of a conventional immersion probe, but can extend the examination of the medium to several different components. This enables in particular the use of two different membranes for sampling. 
     This provides a new possibility to continuously take e.g., cell-free sample from a disposable bioreactor and feed it to an analyzer. This is e.g., also applicable to RM systems, i.e., bioreactors in which the disposable bioreactors are mixed using a rocking motion. 
     The device can also be used for purification, e.g., be designed as a tubular cleaning module. 
     With the device, it is possible to determine the concentration of nutrients and metabolites (such as glucose, lactate, glutamine, glutamate, biogenic amino acids, glycerol, acetate, ethanol or methanol) as well as target proteins (such as monoclonal antibodies, hormones, growth factors, interleukins, interferons) online in a disposable bioreactor at the same time. 
     In one embodiment, the device has only a single membrane, by means of which samples can be taken. Here, the membrane can be designed such that e.g., samples with at least two different particle diameters can be taken from the medium. 
     According to one embodiment, each of the at least two membranes is arranged on an outer region of the extraction region, so that each of the at least two membranes can be arranged in (direct) touch-contact with the medium inside the bioreactor. In this case, the two membranes can be in touch-contact with the medium in particular simultaneously, namely in the operating position of the device. In other words, the extraction region is delimited by two of the two membranes at two different points of its outer region, which is in touch-contact with the medium. Here, each of the membranes is arranged such that it allows in each case at least one component of the medium to pass through the membrane into the interior of the sampling module, e.g., in a transport line of the sampling module. 
     According to one embodiment, the at least two membranes differ at least in that they have differently sized cutoff pore sizes. The cutoff pore size can e.g., be expressed in kDa (kilodaltons) and determines a specific particle size. Components of the medium with a specific particle size smaller than the specific particle size determined by the cutoff pore size can diffuse through the respective membrane. If the particle size of the components deviates too much upward from the cutoff pore size, the particles cannot diffuse through the membrane, or only to a very limited extent. 
     According to a development of this embodiment, a first membrane of the at least two membranes has a cutoff pore size of 50 kDa to 200 kDa, and a second membrane of the at least two membranes has a cutoff pore size of 10 kDa to 30 kDa. The second membrane with the smaller cutoff pore size is particularly suitable for determining the concentration of smaller molecules such as nutrients (e.g., glucose or glutamine) and metabolites (e,g., lactate or glutamate), The first membrane with the larger cutoff pore size is particularly suitable for determining the concentration of larger molecules such as at least one predetermined target protein (e.g., an antibody). 
     According to one embodiment, a first membrane of the at least two membranes is designed and arranged to allow proteins to pass from the medium into the sampling module in touch-contact with the medium. A second membrane of the at least two membranes is designed and arranged to allow nutrients and/or metabolites to pass from the medium into the sampling module in touch-contact with the medium. For example, these can be membranes with the cutoff pore sizes mentioned above. When these membranes are used, two different components of the medium can be determined and/or examined with one device. 
     According to one embodiment, the sampling module has at least one transport line for transporting an adsorber through the extraction region along the transport line. Here, each of the at least two membranes limits the transport line to the outside. In other words, each of the two membranes is arranged between the transport lines and an outer region of the extraction region. The medium to be examined is also arranged in this operating region in the operating position. The outward limitation thus enables the membranes to come into direct touch-contact with the medium. The transport line is designed as a cavity in which the adsorber is guided. The adsorber is used to hold the samples, which in the operating position can diffuse through the respective membrane into the transport line to the adsorber. Different adsorbers can be provided for the different components. Different adsorbers can be transported through the one or more transport lines, which are each designed as adsorbers for the respective sample to be taken. 
     According to one embodiment, at least two different adsorbers are led through the transport line(s), which are each designed as adsorbers for the different components of the medium to be examined. Diffusion of the different components of the medium through the different membranes is triggered and/or supported with a respective adsorber specific therefor. 
     According to a further development of this embodiment, the device has a control module for controlling an adsorber flow through the at least one transport line of the sampling module. The control module can have a processor and/or be designed as a computer. The control module can in particular control one or more valves and/or pumps in such a way that the adsorber(s) is/are guided through the transport line at a predeterminable and/or variable speed. In this case, the adsorber(s) can be stopped, in particular, for a controllable and/or predeterminable time in order to enable enrichment with the components of the medium to be examined. Then the adsorber(s) enriched with the sample(s) can be carried on, in particular out of the extraction region and the sampling module to an analysis system or an analysis module. 
     According to an additional or alternative development of the embodiment, the sampling module has exactly one transport line through the extraction region, and the at least two membranes are arranged one behind the other in the transport direction through the transport line. In this case, two sections of the transport line that are offset from one another in the transport direction are enriched with the respective components of the medium to be examined before the adsorber(s) thus enriched is/are led out of the sampling module. Thus, only the formation of a single transport line is required in the extraction region, which enables a relatively simple and therefore inexpensive manufacture of the sampling module. 
     Alternatively, the sampling module has at least two transport lines through the extraction region, and the at least two membranes are arranged in different transport lines. Here, a separate transport line can be provided and designed for each component to be examined, and thus for each different membrane. Here, the control of the adsorber(s) can be made simpler, since only one enrichment has to take place in each transport line. Furthermore, only one type of adsorber has to be transported through each transport line. Furthermore, the individual components of the medium can be examined separately, e.g., in different analysis systems and/or different constituting parts of an analysis module. 
     According to one embodiment, the membranes are designed as dialysis membranes and/or semipermeable membranes. Such membranes are particularly suitable for bioreactors, since the device reduces the risk of contamination of the contents of the bioreactor. 
     According to one embodiment, the device has an analysis module for examining a sample of the medium taken by the sampling module. The analysis module is designed as a component of the device. The analysis module can be formed in several parts, in particular with at least two different measurement cells for examining at least two different components of the medium. The at least one sample can be supplied to the analysis module e.g., via a transport line, which is arranged running through the sampling module. 
     In a development of this embodiment, the analysis module is designed to determine a concentration of at least two different components in the medium by examining the sample of the medium taken. In particular, the two concentrations of the two different components that have diffused through the two different membranes can be determined. The device can be designed for online determination of the concentration of two different components of the medium. The different components of the medium can e.g., differ by their average molecular size. 
     One aspect relates to a method for examining a medium inside a bioreactor, in particular a disposable bioreactor, with the steps:
         arranging an extraction region of a sampling module in touch-contact with the medium inside the bioreactor;   taking at least one sample of the medium through two different membranes of the sampling part;   examining the sample of the medium taken.       

     The method can in particular be carried out with a device according to the aspect described above. For this reason, the statements made with regard to the device also relate to the method and vice versa, 
     In particular when examining antibodies or target proteins using certain techniques such as MS or HPLC, it can be useful to increase the concentration of the antibodies even further. The concentration in the adsorber could be too low. Therefore, the method can e.g., be supplemented by a downstream chromatography, especially in the form of a capture step (e.g., Protein A Bind+Elute). 
     With the method, e.g., at least two samples of at least two different components of the medium can be taken. Alternatively, only a single sample can be taken, which is taken chronologically and/or spatially in succession on each of the at least two membranes. In other words, a sample of a first component can first be taken on a first membrane, then this sample can be supplemented on a second (different) membrane. 
     Here, the different components of the medium can diffuse through one of the two different membranes into the sampling module. 
     The method can be carried out online and be repeated (e.g., at regular intervals). With the method, e.g., the concentration of at least two different components of the medium in the bioreactor can be examined, determined and/or monitored. 
     In one embodiment, the two membranes are arranged in touch-contact with the medium in such a way that components of the medium pass through each of the two different membranes into at least one transport line through the extraction region of the sampling module. The at least one transport line runs at least through the extraction region of the sampling module and serves to receive and/or to transport the at least one sample. 
     In a development of the embodiment, an adsorber flow through the at least one transport line is controlled such that the components of the medium pass through each of the membranes into an adsorber resting in the transport line for a predeterminable period of time before the enriched adsorber is transported to an analysis module along the at least one transport line. 
     In one embodiment, the sample of the medium has at least two components that are extracted separately from one another through the two different membranes by an air bubble. In general, the components of the sample can be extracted spatially and/or temporally separated from one another through the two membranes. A first component of the sample taken through the first membrane may also be referred to as a first sample and/or first fraction and e.g., have at least a first type of sample particle. A second component of the sample taken through the second membrane may also be referred to as a second sample and/or second fraction and e.g., have at least a second type of sample particle. The two fractions are taken at least spatially separate from one another. The two fractions can e.g., be separated from each other by an air bubble. The air bubble can be generated by specifically controlling the fluids contained in the transport lines. 
     One aspect relates to the use of a device for examining a medium inside a bioreactor according to the above-described aspect for carrying out the method for examining a medium inside a bioreactor according to the further aspect described above. 
     In the context of this invention, the terms “substantially” and/or “approximately” can be used to include a deviation of up to 5% from a numerical value following the term, a deviation of up to 5° from a direction following the term and/or from an angle following the term. 
     Terms such as top, bottom, above, below, etc.—unless otherwise specified—refer to the Earth&#39;s reference system in an operating position of the subject matter of the invention. 
    
    
     
       The invention will be described in more detail below with reference to embodiments shown in the figures. The same or similar reference numerals can designate the same or similar features of the embodiments. Individual features shown in the figures can be implemented in other exemplary embodiments. The figures show: 
         FIG. 1A  a schematic representation of a first embodiment of an extraction region of a sampling module with a common transport line; 
         FIG. 1B  a schematic representation of a second embodiment of an extraction region of a sampling module with two separate transport lines; 
         FIG. 1C  a schematic representation of a third embodiment of an extraction region of a sampling module with a partly separate and partly common transport line; 
         FIG. 2  a schematic representation of the operating principle of the sampling; 
         FIG. 3  a schematic representation of a device for examining a medium inside a disposable bioreactor; 
         FIG. 4  a schematic representation of a device for examining a medium inside a disposable bioreactor with a serial sampling module; 
         FIG. 5  a schematic representation of a device for examining a medium inside a disposable bioreactor with a parallel sampling module; 
         FIG. 6  a schematic flow chart for controlling a device for examining a medium inside a disposable bioreactor with a serial sampling module; and 
         FIG. 7  a schematic flow chart for controlling a device for examining a medium inside a disposable bioreactor with a parallel sampling module. 
     
    
    
     A device for examining a medium inside a bioreactor has at least one sampling module and can also have an analysis module. 
     A sampling module can have at least one pipeline as a transport line. The pipeline can be substantially U-shaped, terminated, fluid-transporting and/or made of plastic. In particular, the transport line can be formed in two parts such that two opposite transport directions are provided inside the transport line, i.e., for example a delivery and a removal line. 
     The sampling module has an extraction region that can be designed as a section of each pipeline in contact with the medium. The extraction region is designed and provided to be inserted into the interior of the bioreactor through the reactor wall of the bioreactor in such a way that the extraction region is in direct touch-contact with the medium. 
       FIG. 1A  shows a schematic representation of a first embodiment of an extraction region  10  of a sampling module with a common transport line  14 . 
     At least one membrane, in particular a dialysis membrane, is arranged in the extraction region  10 . In the embodiment shown, the extraction region  10  has a first membrane  15  and a second membrane  16 , which are arranged in series in the transport direction T. The membranes  15  and  16  are each arranged in a recess in an outer wall of the transport line  14  in such a way that they at least partially seal the transport line  14  at this recess. At the respective recess, the respective membrane  15 ,  16  replaces the recessed line wall of the transport line  14 . Since the membranes  15  and  16  are at least partially permeable to predetermined components of the medium, the seal is not completely but only partially formed. 
     The transport direction T is marked with arrows in the figures and follows the course of the common transport line  14  through the extraction region  10 . The transport direction T runs through a common detection channel  12  (as a delivery line) of the transport line  14  to a probe tip  11 , at which the common detection channel  12  terminates. On the probe tip  11 , the transport line  14  is substantially U-shaped, which is why the transport direction T is reversed here by approximately 180°. In other words, the transport direction T reverses at the probe tip  11 . 
     After this turn at the probe tip  11 , the transport direction T runs from the probe tip  11  along a common removal channel  13 . The common detection channel  12  is arranged substantially parallel to the common removal channel  13 , the directions of transport T through these two channels  12  and  13  to one another essentially being opposite. 
     The inlet and outlet of the transport line  14  can be terminated with a pipe connection element at the probe tip  11 . 
     Different membranes are combined with one another in the transport line  14 , namely the first membrane  15  and the second membrane  16 . These membranes  15 ,  16  can be designed as semipermeable membranes and/or dialysis membranes. The two membranes  15 ,  16  can have different properties, in particular different cutoff pore sizes. For this reason, the two membranes  15 ,  16  hold back and/or allow different components of a medium of a bioreactor to pass through. For example, the second membrane  16  can e.g., have a cutoff pore size of about 10 kDa to about 30 kDa, which only allows smaller molecules such as nutrients (e.g., glucose) and metabolites (e.g., lactate) to pass through the second membrane  16 . The first membrane  15  can e.g., have a cutoff pore size of about 50 kDa to about 200 kDa, which allows larger components (e.g., a target protein such as an antibody of the medium) to pass through the first membrane  15 . 
     An advantage of the two membranes connected in series can be that in a first fraction, which is assigned to the first membrane, a substantially protein-free sample for e.g., a nutrient and/or metabolite analysis may be included. This first fraction can be spatially and/or temporally from a second fraction assigned to the second membrane. In the second fraction, e.g., a protein and various other components such as nutrients, metabolites etc. may be present. It can happen that any existing nutrients do not interfere with the analysis and/or purification of the proteins. With the first fraction, the proteins and antibodies would be quite disturbing for e.g., an enzymatic analysis. The first and second fractions can correspond to first and second samples, respectively, which can be extracted through the respective membrane. 
     In one embodiment, the second membrane can be formed as a 10 kDa Hydrosart membrane, e.g., the Sartorius type: 14439. In this or another embodiment, the first membrane can be designed as a 100 kDa Hydrosart membrane, e.g., the Sartorius type: 14468. 
     Alternatively, another material can be used, so that instead of the Hydrosart (a cross-linked cellulose) e.g., polyethersulfone can be used, corresponding to Sartorius types 14639 and 14668. 
     Furthermore,
         the Ultracel series (RC membrane from Millipore, cellulose based) and/or   the omega series (PES UF from Pall) and/or   hollow fibers, e.g., made of polysulfone from spectrum       

     can also be used as membranes. 
     Through the two membranes  15  and  16 , different components of the medium can pass into the same, common transport channel  14  at positions offset serially one behind the other along the transport channel  14 . One or more adsorber solution(s) can be provided in the transport channel  14  as a means of transport. The adsorber solution can be designed and/or provided for receiving the components of the medium in the transport channel  14  and for transporting the components received along the transport channel  14 . The different components of the medium, which pass through the two different membranes  15 ,  16  into the same transport channel  14 , can be transported along the same, common transport channel  14  past the probe tip  11  out of the extraction region  10  and the bioreactor. 
       FIG. 1B  shows a schematic representation of a second embodiment of an extraction region  10   a  of a sampling module with two separate transport lines  14 . Overall, the second embodiment is similar to the first embodiment, which is why similar and/or the same features of the embodiments are designated with the same reference numerals. 
     In contrast to the first embodiment, the extraction region  10   a  shown in  FIG. 1B  has two separate transport lines  14 . A first one of these separate transport lines  14  has a first detection channel  12   a,  which is deflected at the probe tip  11  to a first removal channel  13   a.  The first membrane  15  is integrated in an outer wall of the first detection channel  12   a.  First components of the medium can pass through the first membrane  15  into the first detection channel  12   a  and can be transported out of the extraction region  10   a  and out of the sampling module through the first removal channel  13   a.    
     A second one of the separate transport lines  14  has a second detection channel  12   b,  which is deflected at the probe tip  11  to a second removal channel  13   b.  The second membrane  16  is integrated in an outer wall of the second detection channel  12   b.  Second components of the medium can pass through the second membrane  16  into the second detection channel  12   b  and can be transported out of the extraction region  10   a  and out of the sampling module through the second removal channel  13   b  separately from the first components. 
       FIG. 1C  shows a schematic representation of a third embodiment of an extraction region  10   b  of a sampling module with partly separate transport lines  14  and partly common transport lines  14 . Overall, the third embodiment is similar to the first and second embodiments, which is why similar and/or the same features of the embodiments are designated with the same reference numerals. 
     In contrast to the second embodiment, the extraction region  10   b  shown in  FIG. 1C  has two transport lines  14  which are only partially separated from one another. A first detection channel  12   a  is deflected at the probe tip  11  to a common removal channel  13 . The first membrane  15  is integrated in an outer wall of the first detection channel  12   a.  First components of the medium can pass through the first membrane  15  into the first detection channel  12   a  and can be transported out of the extraction region  10   b  and out of the sampling module through the common removal channel  13 . 
     A second detection channel  12   b  is likewise deflected at the probe tip  11  to the common removal channel  13 . The second membrane  16  is integrated in an outer wall of the second detection channel  12   b.  Second components of the medium can pass through the second membrane  16  into the second detection channel  12   b  and (together with the first components) can be transported out of the extraction region  10   b  and out of the sampling module through the removal channel  13 . 
     The embodiments shown in  FIGS. 1A, 1B and 1C  can be controlled with the transport direction T reversed. 
     Different adsorber liquids for adsorbing and/or transporting the different components of the medium can be guided in the separate detection channels  12   a  and  12   b.    
     In other embodiments not shown in the figures the transport lines can also be designed differently and/or more than two different membranes can be used, e.g., three membranes for examining at least three different components of the medium. 
     The membranes  15  and/or  16  can be fastened to the transport line  14  by means of different joining techniques, which line can e.g., can be formed from plastic. For example, as a technique “heat sealing” can be used, i.e., thermal welding of the membrane to the plastic carrier. In the case of a cellulose-based membrane, for example, this joining technique can be disadvantageous, since this material cannot be easily welded to another plastic. In this case, i.e., when a cellulose-based membrane is used, the following alternatives can be used: 
     a) Gluing: The membrane can be glued with a special adhesive. Due to possible problems with extractables/leachables, this alternative is rather unfavorable in the biotech sector. 
     b) Clamping: The membrane can be clamped and/or pressed between plastic parts, e.g., also using elastomer seals in between. 
     c) Overcasting: Here, parts of the membrane are covered with a thermoset/elastomer (e.g., silicone) for fixing and sealing. 
     d) Overmolding: recasting and/or overmolding of the membrane with a thermoplastic. 
     These joining techniques can also be used with other membranes. 
       FIG. 2  shows a schematic representation of the operating principle of the sampling. Here, emphasis is placed on particular on the sampling by means of a sampling module that has the first extraction region  10  shown in  FIG. 1A  with the membranes  15 ,  16  arranged in series one behind the other. The first extraction region  10  is only partially shown, in particular without the probe tip  11  and without the removal channel  13 . 
     The upper half of  FIG. 2  shows a medium  100  that has several components. Three different components of the medium  100  are represented by different symbols. The medium thus has a plurality of cells  101 , which are shown as large, cross-striped circles. Furthermore, the medium  100  has a plurality of proteins and/or antibodies  102 , which are shown as empty, medium-sized circles. Finally, the medium  100  has a plurality of smaller molecules  103 , which are shown as small dots. The smaller molecules  103  can e.g., be formed as glucose and/or lactate. The cells  101 , the proteins  102  and the smaller molecules  103 , mixed together as a medium  100 , are arranged inside a bioreactor (not shown). 
     The extraction region  10  of a sampling module, which is partially shown in  FIG. 2 , is introduced into the interior of the bioreactor. The extraction region  10  is arranged inside the bioreactor in such a way that at least the two membranes  15  and  16  are arranged in direct touch-contact with the medium  100 . Here, the first membrane  15  is at least partially arranged as a border region between the medium  100  and the interior of the detection channel  12 . At another location on the transport line  14 , the second membrane  16  is at least partially arranged as a border region between the medium  100  and the interior of the detection channel  12 . An intermediate region  17  is formed in the interior of the transport line  14  between the two membranes  15  and  16 . In the transport direction T through the detection channel  12 , first the second membrane  16  is arranged, which adjoins the intermediate region  17 , which in turn adjoins the first membrane  15 . 
     In the embodiment shown, the second membrane  16  has a smaller cutoff pore size than the first membrane  15 , e.g., from 10 kDa to 30 kDa, preferably from 10 kDa to 20 kDa. For this reason, only the smaller molecules  103  can pass through and/or diffuse through the second membrane  16  into the transport line  14 . A second diffusion time granted for this can be predetermined and/or adjustable and/or controllable and/or regulatable. During the second diffusion time, an adsorber located in the transport line  14  may not be driven, but rather left to rest in order to give the smaller molecules  102  time to pass into the transport channel  14 . The adsorber can then be driven a little further, e.g., up to the first membrane  15 . 
     The first membrane  15  has a larger cutoff pore size than the second membrane  16 , e.g., from 50 kDa to 200 kDa, preferably from 75 kDa to 125 kDa, For this reason, the medium-sized components of the medium  100  can diffuse through the first membrane  16 , in particular the proteins and/or antibodies  101 . A first diffusion time granted for this can be predetermined and/or adjustable and/or controllable and/or regulatable. During the first diffusion time, the adsorber located in the transport line  14  may not be driven, but can be left to rest in order to give the medium-sized molecules  102  time to pass into the transport channel  14 . The adsorber can then be driven further. In order to transport all samples of the medium out of the sampling module and to analyze them in an analysis system and/or an analysis module. 
     The adsorber can be controlled via an external drive. In particular, a region with little and/or no concentration can be formed in the intermediate region  17 , e.g., an air bubble as a separating agent between the two detection regions inside the transport line  14 . 
     Alternatively, the adsorber may not be stopped at the first and/or second diffusion time, but can be guided through the transport line  14  substantially at a constant speed. 
     By sampling shown schematically in  FIG. 2 , samples of two different components of the medium  100  can be taken. These samples can be analyzed in an external analysis system and/or an external analysis module. For example, the concentration of the two components in the medium  100  can be detected and/or determined in this way. 
       FIG. 3  shows a schematic representation of a device for examining a medium inside a bioreactor  200  and/or an RM bioreactor  201 . The bioreactor  200  can be arranged in a tank, e.g., a stainless steel tank. The bioreactor itself can be designed as a disposable bioreactor, e.g., as a plastic bag filled with the medium  100 , which is arranged in the tank shown. The bioreactor  200  can be mixed e.g., by means of a stirring mechanism. The stirring mechanism may have a driven axle that projects into the bioreactor  200 . The stirring mechanism can also drive the medium  100  by means of rocking motion (abbreviated as RM), that is to say mix it by means of a rocking motion. Such an RM bioreactor  201  is shown schematically in  FIG. 3  below. 
     The device has a sampling module  20 . The sampling module  20  has at least one extraction region, e.g., the extraction region  10 ,  10   a  and/or  10   b  shown in  FIGS. 1A, 1B and 1C . 
     The sampling module  20  is arranged such that it at least partially penetrates the reactor wall of the bioreactor  200  and/or  201 . In an operating position, the extraction region  10  of the sampling module  20  can in particular be arranged completely inside the bioreactor  200  and/or  201 . Here, in particular, the two membranes  15  and  16  are in direct touch-contact with the medium  100 . 
     The transport line  14  through the extraction region  10  leads into and out of the sampling module  20  and out to an analysis module  30  of the device for examining the medium  100 . A sterile connection  21  can optionally be formed in this transport line  14  arranged externally from the sampling module  20 . Furthermore, a pump  22  can be arranged between the analysis module  30  and the sampling module  20 , e.g., a persistaltic pump for driving a content of the transport line  14  (e.g., an adsorber). 
     The basic structure of an analysis system for measuring nutrients in a bioreactor is already known. In the analysis module shown, however, besides the measurements of the nutrients (such as glucose, lactate, etc., i.e., the smaller molecules  103 ), a measurement of the content of target proteins  102  can also be carried out. 
     The analysis module  30  can have at least one measurement cell and at least one analyte sensor  31  in order to determine the concentration of at least two components of the medium  100 . Sample residues that have already been examined and/or are not required can be disposed of in a waste container  32 . For this purpose, the analysis module  30  can be connected to the waste container  32  via a waste line. 
     Furthermore, the analysis module  30  can be connected to a first calibration solution  33   a  and a second calibration solution  33   b,  which are designed to calibrate the analysis module  30 . An inspection device  34  can be used to inspect the calibration solutions  33   a  and  33   b.    
       FIG. 4  shows a schematic representation of a device for examining a medium  100  inside a bioreactor  200  with a serial sampling module  20  with more details than  FIG. 3 . In particular, additional components and/or features of the analysis module  30  are shown in  FIG. 4 . The serial sampling module  20  may have the extraction region  10  shown in  FIG. 1A , in which the membranes  15  and  16  are arranged in series one behind the other. 
     A transport solution  36  from a tank serves to pass through the transport line  14 . The transport solution  36  can contain a buffer and/or adsorber for receiving predetermined components of the medium  100 . The transport solution  36  is connected to a left inlet of a fifth valve V 5  via a first measuring pump P 1  and a filter F. A right inlet of the valve V 5  is connected to the sampling module  20 , e.g., via the transport line  14 . 
     A partial outlet of the fifth valve V 5  is connected to a left inlet of a fourth valve V 4 . A right inlet of the fourth valve V 4  is connected to a diffusion module  37 . A partial outlet of the fourth valve V 4  is connected to a first measurement cell  35   a.  Moreover, the diffusion module  37  is connected directly to the first measurement cell  35   a.    
     The first measurement cell  35   a  is connected to a left inlet of a third valve V 3 . A right inlet of the third valve V 3  is connected to the sampling module  20 , e.g., via a channel of the transport line  14 . Furthermore, the first measurement cell  35   a  is connected to a waste container  32 , just like the diffusion module  37 . 
     A first calibration solution  33   a  is connected to a left inlet of a second valve V 2 . A second calibration solution  33   b  is connected to a right inlet of the second valve V 2 . A partial outlet of the second valve V 2  is connected to the diffusion module  37  via a third pump P 3 , which can be designed as a sampler pump. 
     A binding buffer  39   a  is connected to a left inlet of a first valve V 1 . A solution buffer  39   b  is connected to a right inlet of the first valve V 1 . A partial outlet of the first valve V 1  is connected to a left inlet of a sixth valve V 6  via a fourth pump P 4 , which can be designed as an adsorber pump, and via a filter F. 
     A right inlet of the sixth valve V 6  is connected to a left inlet of a seventh valve V 7 . A partial outlet of the seventh valve V 7  is connected to the waste container  32 . A right inlet of the seventh valve V 7  is connected to the partial outlet of the third valve V 3 . 
     A partial outlet of the sixth valve V 6  is connected to a second measurement cell  35   b  via a membrane adsorber  38 , said second measurement cell  35   b  being also connected to the waste container  32 . 
       FIG. 5  shows a schematic illustration of a device for examining a medium  100  inside a bioreactor  200  with a parallel sampling module  20 . The parallel sampling module  20  can e.g., have the extraction region  10   a  shown in  FIG. 1B , in which the membranes  15  and  16  are arranged parallel to one another in two separate transport lines  14 .  FIG. 5  shows additional components and/or features of the analysis module  30  of  FIG. 3 . 
     The device shown in  FIG. 5  is largely identical to the device shown in  FIG. 4 . In contrast to the device shown in  FIG. 4 , the transport solution  36  is guided through two separate transport lines  14 . For this reason, the container and/or tank with the transport solution  36  is additionally connected to the sampling module  20 , more precisely to the second transport line  14  through the extraction region  10   a,  via a second pump P 2 , which can be designed as a measuring pump, via a filter F. Here, the transport solution  36  is preferably connected to the first or second detection channel  12   a  or  12   b.  The associated first or second discharge channel  13   a  or  13   b  is connected to the right inlet of the seventh valve V 7  (and above that to the second measurement cell  35   b ). 
     The right inlet of the fifth valve V 5  is directly connected to the other detection channel  12   a  or  12   b,  and the partial outlet of the third valve V 3  is closed. 
     The transport solution  36  can be passed as a buffer from the associated transport buffer container through the second transport line  14  with the membrane, which is permeable to the target protein  102  (the first membrane  15  in the exemplary embodiments shown). The transport solution  36 , which is then enriched with protein  102 , is passed through the seventh valve V 7  and the sixth valve V 6  onto the protein A membrane adsorber  38 . Specific binding of protein A to the Fc part of the antibodies can lead to selective concentration. After a certain time, which can correspond to a certain sample volume, the valve is then switched and an eluate buffer (from the solution buffer  39   b ) then releases the bound antibody from the protein A membrane adsorber  38  again. This eluted antibody is then detected in the second measurement cell  35   b.  This measurement cell  35   b  can be designed as an optical measurement cell, which can be used e.g., for absorption, emission or scatter measurements such as UV/VIS, fluorescence, NIR, MIR, RAMAN, etc. The detection can e.g., be via the absorption in the UV range at 280 nm. In addition, an eluted antibody can be added for special analyzes (e.g., biological automated assays—binding tests, methods such as MS, HPLC etc.). The adsorber is then regenerated again with the binding buffer  39   a  and the cycle can begin again. 
     The method described for the protein A membrane adsorber  38  can also be applied to other modifications of the membrane adsorber  38 , these can be other protein modifications (e.g., protein G, antibody-specific binding structures, receptor proteins) or other chemical modifications of the membrane adsorber (e.g., ion exchange chromatography or hydrophobic interaction). The method step with protein A is optional and can also be omitted. In one embodiment, this can also be measured directly. 
       FIG. 6  shows a schematic flow diagram for controlling a device for examining a medium inside a disposable bioreactor with a serial sampling module  20 , i.e., the device shown in  FIG. 4 , for example. 
     The method is first started in method step  300 . 
     During the subsequent initialization in method step  301 , the components and/or modules of the device can be set to their starting conditions and/or it can be checked whether the components and/or modules of the device are already set to their respective starting conditions. In particular, the starting conditions of the pumps P 1 , P 3 , P 4  and/or valves V 1  to V 7  can be set and/or checked. 
     In the device shown in  FIG. 4 , the following starting conditions are set in method step  301 : the first, second and third valves V 1 , V 2  and V 3  are closed. The fourth and seventh valves V 4  and V 7  are open from inlet to inlet. The fifth valve V 5  is open between the partial outlet and the left inlet. The sixth valve V 6  is open between the partial outlet and the right inlet. All pumps P 1 , P 3  and P 4  are off. 
     The diffusion module  37  is then flushed in method step  302 . To do this, the first measuring pump P 1  is switched on. The fourth and fifth valves V 4  and V 5  were already set during the initialization in such a way that the transport solution  36  now flushes through the diffusion module  37  and, if necessary, flushes any residues into the waste container  32 . 
     The first measurement cell  35   a  is then flushed in method step  303 . For this purpose, the fourth valve V 4  is opened between the partial outlet and the left inlet. Then (with the first measuring pump P 1  still working) the transport solution  36  can be flushed through the first measurement cell  35   a,  with any residues that may be present being flushed into the waste container  32 . 
     The second measurement cell  35   b  is then flushed in method step  304 . For this purpose, the fifth valve V 5  is opened from inlet to inlet and the third valve V 3  is opened between the partial outlet and the right inlet, Then (with the first measuring pump P 1  still working) the transport solution  36  can be flushed through the valves V 5 , V 7  and V 6  through the second measurement cell  35   b,  with any residues that may be present being flushed into the waste container  32 . 
     The first measurement cell  35   a  is then calibrated in method step  305 . For this purpose, the first measuring pump P 1  is first switched on, the fifth valve V 5  is opened from the partial outlet to the left inlet, the fourth valve V 4  is opened from the inlet to the inlet and the second valve V 2  is opened from the partial outlet to the left inlet. A calibration measurement of the first calibration solution  33   a  in the first measurement cell  35   a  is carried out. Then the second valve V 2  is closed and the sample pump P 3  is switched off. 
     Then one waits for a short time. 
     Then, the second valve V 2  is opened from the partial outlet to the right inlet and the sample pump P 3  is switched on. Now, the first calibration solution  33   a  can be pumped to the diffusion module  37  and/or the first measurement cell  35   a.  A further calibration measurement of the first calibration solution  33   a  is carried out in the first measurement cell  35   a.  Then, the second valve V 2  is closed and the sample pump P 3  is switched off. 
     The following method steps  307  and  306  can take place in parallel or alternatively to one another. 
     In method step  306 , the adsorber for the solution buffer  39   a  is prepared. For this purpose, the sixth and first valves V 6  and V 1  are opened from the partial outlet to the left inlet and the adsorber pump P 4  is turned on. Now, the solution buffer  39   a  is pumped through the second measurement cell  35   b.    
     In method step  307 , a measurement is carried out on the first measurement cell  35   a.  For this purpose, the fifth valve V 5  is opened from inlet to inlet, the fourth valve V 4  is closed, the third valve V 3  is opened from inlet to inlet, and the first pump P 1  is switched off. 
     Then one can wait e.g., for a predetermined diffusion period until components (e.g., the smaller molecules  103 ) of the medium  100  as the first sample have diffused through one of the two membranes  15  and  16  (in the exemplary embodiment shown in  FIG. 2  through the second membrane  16 ). 
     Then the first measuring pump P 1  is switched on until the first sample thus taken is transported into the first measurement cell  35   a.  A measurement takes place in the first measurement cell  35   a,  in particular a determination of the concentration of the respective components of the medium  100  to be examined. 
     The first measuring pump P 1  is then switched off again. 
     After the two method steps  306  and  307 , a switch is made to the second measurement cell  35   b  in method step  308 . For this purpose, the third valve V 3  is opened from the partial outlet to the right inlet, so that solution buffers  39   a  reach the sampling module  20  through the sixth and seventh valves V 6  and V 7 . 
     In method step  309 , a sample of the target protein  102  is taken in the sampling module. For this purpose, the fourth pump P 4  is switched off, the sixth valve V 6  is opened from the partial outlet to the right inlet, the first valve V 1  is closed, and the first measuring pump P 1  is switched on. Then one can wait e.g., for a predetermined diffusion period until components (in particular proteins  102 ) of the medium  100  as a second sample have diffused through one of the two membranes  15  and  16  (in the exemplary embodiment shown in  FIG. 2  through the first membrane  15 ). 
     In method step  310 , a protein measurement is carried out on the second measurement cell  35   b.  Then, the first measuring pump P 1  is switched off (alternatively, the third valve V 3  can also be opened from inlet to inlet for a further measurement on the first measurement cell  35   a  in method step  307 ). 
     This is followed by flushing, the sixth valve V 6  being opened from the inlet to the inlet, the seventh valve V 7  being opened from the partial outlet to the left inlet, the first valve V 1  being opened from the partial outlet to the right inlet, and the fourth pump P 4  being switched on. The solution buffer  39   b  now displaces the binding buffer  39   a.  Both dilution and measurement take place in the second measurement cell  35   b,  the sixth valve V 6  being opened from the partial outlet to the left inlet. The binding buffer  39   a  transports the antibody to the membrane adsorber and at the same time ensures good binding of the antibody to the protein of the adsorber, which e.g., can also be a classic prot. A pillar. This can also be done in several cycles until the adsorber is sufficiently loaded. Then, the solution buffer  39   b,  which can be designed as an elute buffer, ensures that the antibody is removed from the protein A again and the highly concentrated antibody solution is transported into the measurement cell. In the measurement in the second measurement cell  35   b,  in particular a concentration determination of the respective components of the medium  100  to be examined (here: the target protein  102 ) can take place. 
     The method terminates with the last method step  311 . Alternatively, the method can also be carried out iteratively, that is, instead, it can be continued again with method step  300  or  301 , etc. 
       FIG. 7  shows a schematic flow diagram for controlling a device for examining a medium inside a disposable bioreactor with a parallel sampling module  20 , i.e., the device shown in  FIG. 5 , for example. 
     The method is first started in method step  400 . 
     During the subsequent initialization in method step  401 , the components and/or modules of the device can be set to their starting conditions and/or it can be checked whether the components and/or modules of the device are already set to their respective starting conditions. In particular, the starting conditions of the pumps P 1  to P 4  and/or valves V 1  to V 7  can be set and/or checked. 
     In the device shown in  FIG. 5 , the following starting conditions are set in method step  401 : The first, second, third and fourth valves V 1 , V 2 , V 3  and V 4  are closed. The fourth and seventh valves V 4  and V 7  are opened from inlet to inlet. The fifth valve V 5  is opened between the partial outlet and the left inlet. The sixth valve V 6  is opened between the partial outlet and the right inlet. All pumps P 1 , P 3  and P 4  are off. 
     The diffusion module  37  is then flushed in method step  402 . To this end, the first measuring pump P 1  is switched on. The transport solution  36  flushes through the diffusion module  37  and any residues that may be present into the waste container  32 . 
     The following method steps  403  to  406  relate to the measurement on the second measurement cell  35   b  and can take place in parallel or alternatively to the method steps  407  to  409 , which relate to the measurement on the first measurement cell  35   a.    
     In method step  407 , the first measurement cell  35   a  is flushed in this way. For this purpose, the fourth valve V 4  is opened between the partial outlet and the left inlet. Then (with the first measuring pump P 1  still working) the transport solution  36  can be flushed through the first measurement cell  35   a,  with any residues that may be present being flushed into the waste container  32 . 
     In method step  408 , the first measurement cell  35   a  is calibrated. For this purpose, the first measuring pump P 1  is first switched on, the fifth valve V 5  is opened from the partial outlet to the left inlet, the fourth valve V 4  is opened from the inlet to the inlet and the second valve V 2  is opened from the partial outlet to the left inlet. A calibration measurement of the first calibration solution  33   a  in the first measurement cell  35   a  is carried out. Then, the second valve V 2  is closed and the sample pump P 3  is switched off. 
     Then one waits for a short time. 
     Then, the second valve V 2  is opened from the partial outlet to the right inlet and the sample pump P 3  is switched on, Now, the first calibration solution  33   a  can be pumped to the diffusion module  37  and/or the first measurement cell  35   a.  A further calibration measurement of the first calibration solution  33   a  is carried out in the first measurement cell  35   a.  Then, the second valve V 2  is closed and the sample pump P 3  is switched off. 
     In method step  409 , a measurement is carried out on the first measurement cell  35   a.  For this purpose, the fifth valve V 5  is opened from inlet to inlet, the fourth valve V 4  is closed, the third valve V 3  is opened from inlet to inlet, and a measuring loop is carried out in which: 
     a) the first pump P 1  is switched off. 
     b) one waits for a predetermined diffusion period until components (e.g., the smaller molecules  103 ) of the medium  100  as the first sample have diffused through one of the two membranes  15  and  16  (in the exemplary embodiment shown in  FIG. 2  through the second membrane  16 ). 
     c) then, the first measuring pump P 1  is switched off again. 
     d) the first measuring pump P 1  is switched on until the first sample thus taken is transported into the first measurement cell  35   a.  A measurement takes place in the first measurement cell  35   a,  in particular a determination of the concentration of the respective components of the medium  100  to be examined. 
     e) one waits again for a short time. 
     In parallel or as an alternative to these steps, the second measurement cell  35   b  is flushed in method step  403 . For this purpose, the second measuring pump P 2  is switched on, as a result of which the transport solution  36  is flushed through the valves V 7  and V 6  and through the second measurement cell  35   b,  with any residues that may be present being flushed into the waste container  32 . 
     In method step  404 , the adsorber for the solution buffer  39   a  is prepared. To this end, the second measuring pump is switched off, the sixth and first valves V 6  and V 1  are opened from the partial outlet to the left inlet, and the adsorber pump P 4  is switched on. The solution buffer  39   a  is now pumped through the second measurement cell  35   b.    
     In method step  405 , a sample of the target protein  102  is taken in the sampling module  20 . For this purpose, the fourth pump P 4  is switched off, the sixth valve V 6  is opened from the partial outlet to the right inlet, the first valve V 1  is closed, and the second measuring pump P 2  is switched on. Then, e.g., one can wait for a predetermined diffusion period until components (in particular proteins  102 ) of the medium  100  as a second sample have diffused through one of the two membranes  15  and  16  (in the exemplary embodiment shown in  FIG. 2  through the first membrane  15 ). 
     The second measuring pump P 2  is switched on again and a diffusion into the second measurement cell  35  is awaited. Then, the second measuring pump P 2  is switched on again. 
     Method step  405  can run in several cycles. 
     In method step  406 , a protein measurement is carried out on the second measurement cell  35   b.  First, the second measuring pump P 2  is switched off. 
     This is followed by flushing, wherein the sixth valve V 6  is opened from the inlet to the inlet, the seventh valve V 7  is opened from the partial outlet to the left inlet, the first valve V 1  is opened from the partial outlet to the right inlet, and the fourth pump P 4  is switched on. The solution buffer  39   b  now displaces the binding buffer  39   a.  Both dilution and measurement take place in the second measurement cell  35   b,  wherein the sixth valve V 6  is opened from the partial outlet to the left inlet. During the measurement in the second measurement cell  35   b,  in particular a concentration determination of the respective components of the medium  100  to be examined (here: the target protein  102 ) can take place. 
     The method is terminated with the last method step  410 . Alternatively, the method can also be carried out iteratively, that is, instead, it can be continued again with method steps  400  or  301  etc. 
     The method steps highlighted in gray in  FIGS. 6 and 7  are necessary method steps, while some or all of the method steps highlighted in white can be omitted or can only be carried out optionally. 
     REFERENCE NUMERAL LIST 
       10  extraction region 
       10   a  extraction region 
       10   b  extraction region 
       11  sample tip 
       12  common detection channel 
       12   a  first detection channel 
       12   b  second detection channel 
       13  common removal channel 
       13   a  first removal channel 
       13   b  second removal channel 
       14  transport line 
       15  first membrane 
       16  second membrane 
       17  intermediate region 
       20  sampling module 
       21  sterile connection 
       22  pump 
       30  analysis module 
       31  analyte sensor 
       32  waste container 
       33   a  first calibration solution 
       33   b  second calibration solution 
       34  inspection device 
       35   a  first measurement cell 
       35   b  second measurement cell 
       36  transport solution 
       37  diffusion module 
       38  membrane adsorber 
       39   a  binding buffer 
       39   b  solution buffer 
       100  medium 
       101  cell 
       102  protein/antibody 
       103  small molecule 
       200  bioreactor 
       201  RM bioreactor 
     F filter 
     P 1  first measuring pump 
     P 2  second measuring pump 
     P 3  sample pump 
     P 4  adsorber pump 
     V 1  first valve 
     V 2  second valve 
     V 3  third valve 
     V 4  fourth valve 
     V 5  fifth valve 
     V 6  sixth valve 
     V 7  seventh valve 
     T transport direction