Methods for Producing Bioproducts

Abstract

The present invention relates to a method for producing a biological product using a bioprocessing installation (1), the bioprocessing installation (1) comprising an electronic process controller (2) and a bioprocessing unit (3), the bioprocessing unit (3) comprising a vessel (4), a clarification unit (5) comprising a centrifuge (6) and a chromatography unit (8) comprising a chromatograph (10), wherein a cell broth obtained from the vessel (4) is conducted in a liquid flow (9) sequentially through the clarification unit (5) and the chromatography unit (8), the clarification unit (5) comprising the centrifuge (6) being operated in a centrifugation cycle comprising centrifugation steps such as a loading step, a washing step and a draining step, and the chromatography unit (8) comprising the chromatograph (10) being operated in a chromatography cycle comprising chromatographic steps such as an equilibration step, a loading step, a washing step, an elution step and a regeneration step. It is proposed that the particle reduction between the liquid flow section leaving the clarification unit (5) and the liquid flow section leaving the chromatography unit (8) is less than 10% in particle concentration, and that the execution of the steps assigned to the centrifugation cycle and the execution of the steps assigned to the chromatography cycle are at least partially synchronized with each other by the electronic process control device (2) in a synchronization routine (13) based on an assigned synchronization strategy (14).

Description

[Technical field]

[0001] The present invention relates to a method for producing a biological product using a bioprocessing installation according to the preamble of claim 1, an electronic process control device for carrying out said method according to claim 15, a computer program product for an electronic process control device according to claim 17, a computer-readable storage medium on which the computer program product is stored according to claim 18, and a bioprocessing installation with an electronic process control device according to claim 19. [Background technology]

[0002] The term "bioprocess" is used herein to refer to any kind of biotechnological process, in particular a biopharmaceutical process. One example of such a bioprocess is the use of a bioreactor to cultivate microorganisms or mammalian cells under defined conditions, whereupon a cell broth is transferred from the bioreactor to a downstream process.

[0003] The term "bioproduct" is used herein to describe any type of chemical compound produced in such a bioprocess. Examples of such bioproducts are proteins, in particular antibodies, growth factors or hormones, metabolites or any other molecules, as well as cells or their components.

[0004] The method for producing biological products can be applied in various fields of biotechnology. High efficiency in this field has been driven by the increasing demand for biological products such as biopharmaceuticals. Efficiency in this sense does not only relate to the cost-effectiveness of the components used, but also to the controllability of the processes involved. The method relies on an optimal interplay between the clarification of the cell broth of the bioprocess and the purification of the biological product in order to optimize the controllability and time efficiency of the bioprocess.

[0005] On the one hand, chromatographs must generate and maintain high and constant pressure for optimal purification results. On the other hand, chromatographs are generally prone to blockage by solid particles, which adversely affects the performance of the chromatograph and leads to impaired purification results. It is therefore common to install a filter immediately upstream of the chromatograph to prevent blockage of the chromatograph. However, the fluid flows due to a pressure difference from the high-pressure side upstream of the filter to the low-pressure side downstream of the filter. The filter itself is the cause of this pressure drop, also called the filter resistance. This pressure drop occurs when the fluid is subjected to frictional forces caused by the flow resistance as it flows through the tube (DIN ISO 11057:2012-05 Emissionen aus stationaeren Quellen; Pruefverfahren für die die Charakterisierung des Filtrationsverhaltens abreinigbarer Filtermedien (ISO 11057:2011); Beuth Verlag, Berlin. p. 9.). This filter resistance creates a damping effect, slowing the transmission of fluid flow and fluid pressure. The primary determinants of resistance to fluid flow are the fluid velocity and fluid viscosity through each pipe. Pressure drop increases in proportion to the frictional shear forces within the piping network. These frictional shear forces increase with the number of intervening components, such as filters.

[0006] Oversized particles and / or particle aggregates can form a filter cake (retentate) and block the filter grid, especially when static filtration is used where the filtrate is pulled in the direction of the fluid flow. In some cases, such blockages can even prevent the fluid phase from crossing the filter, known as clogging (Sparks, Trevor; Chase, George (2015). Filters and Filtration Handbook(6th ed.)). As a result, the concentration of potentially blocking solid particles and / or particle aggregates increases over time due to accumulation on the filter grid. As a result, the filter resistance increases continuously with time. Such pressure changes have been found to lead to impaired processing time, chromatographic performance and service life. Furthermore, any changes in fluid pressure lead to uneven operating demands on the chromatographic stationary phase, which results in uneven separation results with limited reproducibility. Finally, pressure fluctuations can lead to disturbances in sensor data acquisition, which in turn can impair chromatographic analytical results and quality control.

[0007] Alternatively, dynamic filtration can be used, in which the filtrate is pulled perpendicular to the direction of fluid flow. This creates shear stresses that at least limit the formation of a filter cake. As for static filtration, the driving force is also the pressure difference between the high-pressure side and the low-pressure side of the filter, the so-called transmembrane pressure (Handbook of Membrane Separations, Edited by Anil K.Pabby, Syed SHRizvi, Ana aria Sastre, CRC Press). However, even dynamic filtration, such as tangential flow filtration or cross-flow filtration, cannot completely prevent filter clogging. Another problem that occurs during dynamic filtration is that during the process, the transmembrane pressure can decrease due to the increase in filtrate viscosity caused by the continuous concentration of the filtrate. Thus, the filtration efficiency decreases and may take longer in large-scale processes. A further disadvantage is the low energy efficiency of tangential flow filtration, since a large part of the energy introduced during the conveying of the feed stream is lost via the retentate (Rautenbach, Robert: Membranverfahren Grundlagen der Modul- und Anlagenauslegung, Springer-Verlag, 1997).

[0008] One possible way to offset the increased filter resistance would be to increase the fluid flow, however, in bioprocesses using cells for bioproducts, the fluid flow cannot be increased arbitrarily due to increased shear forces that could damage the cells and thus reduce the overall productivity of the process.

[0009] The known method for producing a biological product (WO 02 / 086135), which is the starting point of the present invention, utilizes a bioprocessing installation having an electronic process control device, a bioprocessing unit with a vessel, a clarification unit with a centrifuge, and a chromatography unit with a chromatograph. In this method for producing a biological product, a cell broth is obtained from the bioprocessing unit, which is led through a clarification unit to clarify at least a portion of the cells from the bioprocessing unit. After clarification is completed, the resulting product-containing supernatant liquid is passed through a filter to further remove solid particles, which may extend the service life of the chromatography unit. Finally, the supernatant liquid may be passed through a chromatography unit to purify the biological product. As a result, a combination of limited efficiency occurs in terms of process time, process flexibility, and adjustability in the mechanical settings as well as the controllability of the entire process. Summary of the Invention [Problem to be solved by the invention]

[0010] The object of the present invention is to provide a method for producing a biological product, which increases the efficiency and reproducibility of the bioprocess with as little effort as possible. [Means for solving the problem]

[0011] The above-mentioned problem is solved by a method for producing a biological product having the features recited in the preamble of claim 1 by the features recited in the characterizing part of claim 1.

[0012] The general concept underlying the present invention is based on a direct connection between a clarification unit and a chromatography unit, in which there is little solid-liquid separation, preferably no intervening solid-liquid separation unit, in particular a filtration unit, which allows synchronization of both units.

[0013] The term "solid-liquid separation" as used herein refers to a process that uses various physical and chemical properties of solid or liquid chemicals mixed in solution to separate those chemicals.

[0014] The above is facilitated by removing the intervening solid-liquid separation unit between the clarification unit and the chromatography unit of the bioprocessing equipment. Thus, the disruptive pressure drop caused by the filter can be substantially avoided, thereby providing a basis for constant and delay-free fluid flow and / or fluid pressure. Interestingly, it has been found that this allows the clarification unit and the chromatography unit to be synchronized, thereby even offsetting the absence of the intervening solid-liquid separation unit. Firstly, this reduces waiting and processing times, thus resulting in improved bioprocessing efficiency with less effort. Secondly, it increases controllability and time efficiency, which allows not only cost efficiency but also simplified and more efficient control of the entire process.

[0015] However, it has been found that simply excluding the above-mentioned solid-liquid separation unit is not sufficient, since if no further measures are taken, this may cause blockage of the chromatography column during operation. It is therefore proposed that the chromatography unit is able to pass most of the solid particles, so that these solid particles do not lead to blockage of the chromatography column.

[0016] In particular, it is proposed that the particle reduction between the liquid flow section leaving the clarification unit and the liquid flow section leaving the chromatography unit is less than 10% in particle concentration, preferably less than 5%, and that the execution of the steps assigned to the centrifugation cycle and the execution of the steps assigned to the chromatography cycle are at least partially synchronized with each other by an electronic process control device in a synchronization routine based on an assigned synchronization strategy.

[0017] The term "particle reduction" refers to any reduction in the concentration of solid particles in a suspension, such as cells in a culture medium.

[0018] Claims 2 to 4 relate to preferred embodiments of the connection between the clarification unit and the chromatography unit. These measures solve the above-mentioned problem of disruptive pressure drops caused by any intervening components, such as filters, which damp and / or delay in time the fluid flow as well as the fluid pressure transmission, and form the basis for the synchronization of the clarification unit and the chromatography unit. Furthermore, the direct connection between these units, without any intervening components, basically allows the control of the chromatography unit by the clarification unit, in particular by the centrifuge.

[0019] The preferred embodiments of claims 5 to 8 relate to various synchronization options which allow increased flexibility regarding the selection of the respective synchronization strategy.

[0020] Particularly preferred embodiments for each step to be synchronized are specified in claims 9 to 11. These specifications allow optimal synchronization of the clarification unit and the chromatography unit. This is particularly advantageous since buffers and / or washing liquids can be used together by both units involved. This minimizes redundancies and saves resources such as buffers or culture medium. Another important advantage is that the product-containing supernatant liquid can be guided first to a centrifuge and then to the chromatography unit according to liquid synchronization, in which case this supernatant liquid is directly filled into the chromatograph. In a similar manner, a washing buffer can be used for both washing steps. This can be done on the one hand to rinse the cells with fresh buffer or growth medium and on the other hand to wash out solid particles from the chromatograph. The fresh buffer helps to keep the cells healthy, while the washing out of solid particles increases the service life of the chromatography unit. Additionally, washing out the solid particles solves the problem of particulate build-up discussed above, which would otherwise cause an undesirable increase in pressure drop over time.

[0021] A preferred embodiment according to claim 12 relates to possible differences between the first and second culture environment, such an embodiment allows for increased flexibility regarding the choice of entities, liquid properties and / or culture environment conditions of the first and second culture environment, which is particularly advantageous for adjusting the requirements of the cells and thus allowing the reuse of the cells for subsequent biological products.

[0022] Claims 13 to 15 relate to preferred embodiments of the chromatography unit and the bioprocessing installation. These measures minimize the risk of blockage of the chromatography column by solid particles and / or solid particle agglomerates and provide flexibility regarding the type of chromatographic separation technique.

[0023] According to the second independent teaching of claim 16, the entire bioprocessing installation with the proposed electronic process control device (claim 17) is claimed as such. All statements relating to the proposed method are fully applicable to the proposed bioprocessing installation.

[0024] The third independent teaching according to claim 18 relates to an electronic process control device. The electronic process control device is designed to implement the proposed method according to any of the preceding claims. According to claim 19, the electronic process control device comprises a data processing system for implementing the proposed method, in particular the synchronization routine. All statements relating to the first teaching are fully applicable to this third teaching.

[0025] According to another teaching of claim 20 of equal importance, a computer program product for the proposed data processing system is claimed as such. The computer program product is configured for implementing the proposed method, in particular for implementing the above-mentioned routines. Again, all statements relating to the proposed method are fully applicable to the proposed computer program product.

[0026] According to another teaching of claim 21, which is of equal importance, a computer-readable storage medium on which a computer program product is stored is claimed as such, again all statements relating to the proposed method being fully applicable to the proposed readable storage medium.

[0027] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic diagram showing a preferred embodiment of the proposed bioprocessing facility, capable of carrying out the proposed method. [Diagram 2] 2 is a flow chart showing the working principle of the method proposed by FIG. 1; [Diagram 3] FIG. 2 is a schematic diagram showing preferred synchronization options for the start and / or end of each of the steps to be synchronized. [Figure 4] 1 is a table showing different preferred synchronization options for each step to be synchronized according to the proposed method. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The proposed method for producing a biological product using a bioprocessing installation 1 is preferably assigned to the upstream and downstream steps of a bioprocess for processing liquids in the form of cell broths for cell culture and / or biological production.

[0030] The term "liquid" should be understood in a broad sense: it includes not only pure liquids per se, but also emulsions and suspensions, e.g. heterogeneous mixtures of at least two different liquids or heterogeneous mixtures of solid particles and liquids.

[0031] The term "cell broth" refers to a suspension of cells and / or cell debris in a medium, and represents the culture medium and the whole of each organism cultured in the culture medium. Thus, the term "cell broth source" refers to any manufactured device or system capable of producing and / or storing cell broth.

[0032] The term "upstream processes" includes all steps related to cell bank, inoculum (seed train) development, medium development, growth kinetics optimization and the cultivation process itself, as well as the corresponding in-process controls. Cell harvesting can be considered both as part of the upstream process and as part of the downstream process.

[0033] The term "downstream processing" includes all steps associated with the recovery and purification of biosynthetic products, particularly biopharmaceuticals, from natural sources such as animal or plant tissues or cell broths, including the reuse of recoverable components and the appropriate treatment and disposal of waste products.

[0034] In general, cell culture is currently used for the production of biopharmaceuticals, especially proteins, such as human insulin, growth factors, hormones, vaccines or antibodies, antibody derivatives, etc. The product may also be a non-biopharmaceutical, such as an enzyme for food processing, an enzyme for laundry detergent, a biodegradable plastic, or a biofuel. The focus of the present invention is on biopharmaceutical products, such as antibodies or exosomes, secreted by cells into the supernatant. Additionally or alternatively, the product may be the cells themselves, especially mammalian cells, including stem cells or immune cells such as CAR-T cells for the treatment of cancer.

[0035] As shown in Figures 1 to 4, according to all embodiments the proposed method for producing a biological product using a bioprocessing installation 1 employs at least one electronic process control device 2 and a bioprocessing unit 3. The bioprocessing unit 3 comprises a cell broth source with at least one container 4 for a cell broth comprising a culture medium and cells. Here preferably the at least one container 4 is designed as a bioreactor (Figure 1).

[0036] The term "culture medium" means that the cells used in the bioprocess, preferably microbial or eukaryotic, are grown in a specially designed growth medium that supplies the nutrients required by the respective organism or cell. Various media exist, but always include at least a carbon source, a nitrogen source, water, salts and micronutrients.

[0037] The term "bioreactor" in this context means any manufactured device or system that supports a biologically active environment by allowing for the monitoring and control of at least one parameter.

[0038] According to a preferred embodiment, the bioprocessing unit 3 additionally comprises a second vessel which may be designed as a passive vessel.

[0039] A "passive vessel" refers to a vessel that does not have supply lines and / or does not have electronic process controllers that can monitor at least one parameter within the passive vessel, whereas an "active vessel" refers to a vessel, such as a bioreactor, that has supply lines and / or has electronic process controllers and thus can monitor and / or control at least one parameter within the active vessel.

[0040] According to FIG. 1, the bioprocessing unit 3 includes a clarification unit 5 equipped with a centrifuge 6 for centrifuging the cell broth.

[0041] "Centrifugation" is a term used to describe the settling of particles in an artificially created gravitational field by centrifugal force, whereby significant reductions in separation time are achieved by the use of large acceleration forces.

[0042] Preferably here, the centrifuge 6 is designed as a fluidized bed centrifuge for carrying out a continuous centrifugation process. A preferred set-up of the centrifuge 6 is described in EP 2 485 846 A1, the contents of which are incorporated herein by reference.

[0043] The centrifuge 6 has a rotor which may be rotated about a centrifuge rotor axis by a motor, preferably electric. The centrifuge rotation speed and liquid pumping speed are adjustable by the electronic process controller 2 with the goal of establishing a fluidized bed of particles, such as cells or cell debris, within the centrifuge 6. A fluidized bed is achieved when the centrifugal force on the particles is equal to the opposing fluid force, and therefore there is zero net force on the particles.

[0044] According to Figure 2, the cell broth obtained from at least one vessel 4 is conducted through a clarification unit 5. The clarification unit 5, which comprises a centrifuge 6, is operated in a centrifugation cycle, which comprises centrifugation steps such as a fill step, a wash step and a drain step.

[0045] The centrifuge 6 can be operated in forward operation for cell separation and / or cell washing. "Forward operation" means one of the two possible fluid flow directions in the centrifuge 6 and denotes an operation leading to the separation of liquid and solid particles, such as culture medium and cells. Such "forward operation" allows, on the one hand, the washing of the separated cells with a buffer, preferably a PBS buffer, or a culture medium, preferably a culture medium, more preferably a concentrated culture medium, coming from the buffer / culture medium container 7, and / or, on the other hand, the clarification of the cell broth. The aim here is to clarify the liquid supernatant from solid particles, such as cells, cell debris, etc., which are considered as biomass. The product obtained in this forward operation is a cell broth supernatant containing the product of interest, such as a recombinant protein, in particular an antibody.

[0046] The term "enriched medium" refers to a medium that contains higher concentrations of vitamins, growth factors, carbon sources, nitrogen sources and / or amino acid concentrations, etc., and preferably allows each organism to grow at its maximum growth rate with optimized nutrient concentrations. Growth factors and micronutrients are included in the medium for microorganisms that cannot produce all of the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and cobalt, are usually present in unrefined carbon and nitrogen sources, but may need to be added when using refined carbon and nitrogen sources.

[0047] The filling step refers to the step of filling each centrifugation chamber with the cell broth to be centrifuged obtained from the bioprocessing unit 3 in the forward running of the centrifuge 6 .

[0048] The washing step refers to the washing of each centrifugation chamber with buffer or medium from the buffer / medium container 7 in the forward run of the centrifuge 6. This washing step preferably serves to supply fresh nutrients to the cells.

[0049] Alternatively, the centrifuge 6 can be operated in reverse operation. By "reverse operation" is meant the second of the two fluid flow directions possible in the centrifuge 6 and refers to the operation leading to the discharge of separated solid particles, preferably cells. The product obtained in reverse operation is cells in a cell broth.

[0050] The discharge step thus means the step assigned to the centrifuge 6, in which solid particles, preferably cells, are discharged from the centrifugation chamber in a retraction operation. This discharge step may serve, inter alia, to return the cells to the vessel 4 for reuse of the cells in a subsequent bioprocess.

[0051] As can be seen in Figure 2, the cell broth obtained from the bioprocessing unit 3 is conducted in liquid stream 9 sequentially through a clarification unit 5 and then through a chromatography unit 8. The chromatography unit 8 further comprises at least one chromatograph 10, at least one buffer reservoir 11 and at least one elution reservoir 12. The chromatography unit 8 including the chromatograph 10 is operated in a chromatography cycle including chromatography steps such as an equilibration step, a loading step, a washing step, an elution step and a regeneration step.

[0052] The equilibration step refers to the step where the system enters an equilibrium state. In the case of the chromatography unit 8, this refers to filling each chromatograph 10 with the respective buffer to be used in the subsequent purification of the bioproduct until its entire volume is occupied by the respective buffer.

[0053] The loading step refers to the step of loading the product-containing supernatant obtained from the centrifuge 6 into each chromatograph 10, thereby binding the product to the chromatograph 10 and purifying the bioproduct in a subsequent purification step by the chromatography unit 8.

[0054] A wash step refers to washing the chromatograph 10 with a buffer or medium from a buffer reservoir 11. This wash step is typically performed to flush the chromatograph 10 to remove non-specifically bound compounds.

[0055] The elution step refers to the extraction of one material from another, for example, by washing with a solvent such as water or imidazole. As used herein, the elution step refers to the extraction of the bioproduct from each chromatograph 10 using an aqueous solution having a pH gradient and / or a conductivity gradient.

[0056] A regeneration step refers to a step of restoring a material or a system. In this particular context, a regeneration step refers to the re-equilibration and / or cleaning of the chromatograph 10, for example with a NaOH solution.

[0057] Of particular importance to the present invention is that the particle reduction between the liquid stream section leaving the clarification unit 5 and the liquid stream section leaving the chromatography unit 8 is less than 10%, preferably less than 5%, more preferably less than 2% in particle concentration. Hence, the reduction of residual particles in the liquid stream 9 is less than 10% between the liquid stream section leaving the clarification unit 5 and the liquid stream section leaving the chromatography unit 8. A preferred embodiment includes a special configuration of the chromatography unit 8, which makes the chromatography unit 8 less prone to clogging and thus able to deal with particles and / or particle agglomerates. Also preferably, the chromatography unit 8 is set up for washing away solid particles and / or particle agglomerates.

[0058] According to the invention, the execution of the centrifugation step and the execution of the chromatography step are at least partially synchronized with one another, which is controlled by the electronic process control device 2 and organized in synchronization routines 13. These synchronization routines 13 are based on assigned synchronization strategies 14.

[0059] The term "synchronization" refers to the two-way timing coordination of events to make systems operate simultaneously.

[0060] In the embodiment according to FIGS. 1 and 2, here preferably the chromatography unit 8 receives the liquid flow fraction leaving the clarification unit 5 in an unfiltered state.

[0061] The term "unfiltered" here means that no separation step of any kind is carried out between the liquid stream fraction leaving the clarification unit 5 and the liquid stream fraction leaving the chromatography unit 8, leading to a solid particle reduction of more than 10%, preferably more than 5%, even more preferably more than 2%. Exemplary separation processes are filtration, in particular dead-end or cross-flow filtration, centrifugation or reverse osmosis.

[0062] In a preferred embodiment, the chromatography unit 8 has a feed inlet directly connected to the outlet of the centrifuge 6. According to this embodiment, no additional devices need to be interposed between the centrifuge 6 and the chromatography unit 8, as can be seen in Figures 1 and 2.

[0063] In another preferred embodiment, the liquid flow 9 is at least temporarily continuous. Thus, the chromatography unit 8, in particular the chromatograph 10, can be controlled by controlling the clarification unit 5, in particular the centrifuge 6.

[0064] By "continuous" it is meant here that the liquid stream 9 from the bioprocessing unit 3, passing through the clarification unit 5 and finally through the chromatography unit 8, is at least temporarily one continuous flow without being stopped or delayed.

[0065] According to another preferred embodiment, as can be seen in Fig. 3, for at least one synchronization routine 13, a synchronization strategy 14 represents a temporal synchronization, whereby at least one centrifugation step remains in a predefined temporal relationship with respect to at least one chromatography step.

[0066] The term "temporal synchronization" as used herein means a bidirectional coordination of events according to a predefined temporal relationship between each centrifugation step and each chromatography step.

[0067] In another embodiment, according to the temporal synchronization, the steps to be synchronized start at the same time, as can be seen in Fig. 3 (Fig. 3a). Additionally or alternatively, the steps to be synchronized end at the same time (Fig. 3b). In yet another embodiment, the steps to be synchronized start and / or end at the same time or start and / or end with a predefined delay (Fig. 3c). The steps to be synchronized can be any centrifugation step or any chromatography step.

[0068] The term "predetermined" here means that it can be defined in advance, preferably by a user.

[0069] According to a particularly preferred embodiment, according to the temporal synchronization, one of the steps to be synchronized is started depending on the start of the other step, respectively. Alternatively, one of the steps to be synchronized is started depending on the end of the other step, respectively (FIG. 3d).

[0070] 4, for at least one synchronization routine 13, the synchronization strategy 14 represents a liquid synchronization, which is preferably performed in addition to a time synchronization. According to the liquid synchronization, a liquid is transferred to the chromatography unit 8 in at least one centrifugation step.

[0071] The term "hydraulic synchronization" here means a bidirectional coordination of events according to a predefined hydraulic relationship between each centrifugation step and each chromatographic step. "Hydraulic" preferably means a fluidic connection between the components involved. As an example, the liquid passing through the clarification unit 5, in particular the centrifuge 6, can be led directly to the chromatography unit 8, in particular the chromatograph 10, to combine one liquid for both units 5, 8. This can be achieved by using separate liquid lines 15 or the same liquid line. The liquid can be a supernatant, a buffer, a growth medium, etc.

[0072] In another particularly preferred embodiment according to Fig. 4, for at least one synchronization routine 13, the loading step of the centrifugation cycle is synchronized with the loading step of the chromatography cycle according to a synchronization strategy 14. It is particularly noteworthy that the product-containing supernatant liquid leaving the centrifuge 6 resulting from the centrifugation in forward operation can be loaded directly onto the chromatograph 10 according to liquid synchronization.

[0073] According to another particularly preferred embodiment according to Fig. 4, for at least one synchronization routine 13, the wash steps of the centrifugation cycle are synchronized with the filling steps of the chromatography cycle according to a synchronization strategy 14. Preferably, the synchronization strategy 14 represents a hydraulic synchronization. This is particularly important since dead volumes are taken into account. This ensures that as much product-containing supernatant liquid as possible is filled into the chromatograph 10.

[0074] Additionally or alternatively, the washing steps of the centrifugation cycle are synchronized with the washing steps of the chromatography cycle. It is particularly important that the liquid used for the washing steps of the centrifugation cycle and leaving the centrifuge 6 can be led directly to the chromatograph 10 to realize the washing steps of the chromatography cycle, designed for washing away non-specifically bound compounds and solid particles. Preferably, the synchronization strategy 14 represents a liquid synchronization. The same liquid can thus be used both for the washing steps of the centrifugation cycle and for the washing steps of the chromatography cycle. The liquid may be any buffer or culture medium etc. that is (bio)compatible with the bioproduct producing cells in the centrifuge 6 on the one hand and with the stationary phase of the chromatography cycle 10 on the other hand. Particularly preferably, at least one liquid is a combined liquid used for both the centrifugation cycle and the chromatography cycle.

[0075] In another embodiment according to Fig. 4, for at least one synchronization routine 13, the drain step of a centrifugation cycle is synchronized with one of the equilibration, washing, elution and / or regeneration steps of a chromatography cycle according to a synchronization strategy 14. Here, preferably, the synchronization strategy represents a time synchronization.

[0076] Here, preferably, according to figures 1 and 2, the cell broth is transferred from a first culture environment A established by the bioprocessing unit 3 to a centrifuge 6. In a retraction operation of the centrifuge 6, at least a part of the discharged cells is transferred into a separate second culture environment B, which is different from the first culture environment A and which is established for a subsequent cell culture and / or biological production.

[0077] Additionally or alternatively, the second culture environment B differs from the first culture environment A in terms of liquid properties, e.g., the selection of the medium, in particular the selection of a concentrated medium, including, inter alia, an optimal carbon source concentration, an optimal nitrogen source concentration, an optimal amino acid concentration and / or an optimal growth factor concentration, and / or the liquid volume used in the bioprocess.

[0078] Additionally or alternatively, the second culture environment B differs from the first culture environment A in terms of culture environment conditions, such as gas concentrations, particularly oxygen and / or carbon dioxide concentration selection, and / or pH selection, and / or temperature selection. Exemplary amino acid sources can be peptone or tryptone at concentrations of 0.5% to 2%. Exemplary carbon sources can be glucose or sucrose at concentrations of 0.1% to 3%.

[0079] It is also preferred that the second culture environment B is the first culture environment A after the culture medium has been exchanged, preferably completely exchanged. In these embodiments, according to Figures 1 and 2, the discharged cells can be transferred for reusing the cells for cell culture and / or biological production after the culture medium and / or cells have been at least partially removed. Thus, the backmixing of the spent culture medium with new culture medium and / or cells can be controlled separately.

[0080] Preferably, the resulting ratio of consumed liquid to new liquid is individually adjustable. Additionally or alternatively, the second culture environment B preferably provides culture environment conditions that are favorable for cell growth and / or biological production. The term "favorable for cell growth and / or biological production" refers to the enhancement and support of at least one parameter that reflects cell viability and / or cell productivity, including, but not limited to, growth rate, viability (i.e., percentage of live cells), productivity, oxygen uptake rate and / or biomass production rate.

[0081] In another preferred embodiment, the second culture environment B can be provided by a container 4, which is rendered cell- and / or liquid-free in a preparation phase in order to establish the second culture environment B. In a preferred embodiment, the preparation phase comprises at least one workflow step for establishing conditions for cell culture and / or biological production.

[0082] Said workflow steps may be at least one of the following groups: medium preparation, (completely) emptying, cleaning, maintaining, sterilizing, replacing and / or filling a container with a medium, preferably a concentrated medium. "Medium conditioning" describes the act of configuring a medium to be used in a bioprocess, in particular the calculation of certain component ratios and the actual production of the medium.

[0083] In a preferred embodiment, the workflow step may be a step of at least partially, preferably completely, emptying the container 4. In another example, the container 4 has to be at least partially, preferably completely, cleaned. In a further example, the workflow step may be a maintenance of the container 4 to ensure an optimal bioprocess, or a replacement of the container 4, especially if the container 4, or especially any container of the bioprocess unit 3, is designed as a disposable device. Another possible workflow step may be the filling of the container 4 with a medium, especially a concentrated medium, as described above. For this procedure, the container first needs to be filled with the medium. Any culture medium can be used, preferably any concentrated medium with a higher nutrient concentration, etc. Even more preferably, these media do not constitute a limiting factor for cell culture and / or biological production. Here, preferably, but merely by way of example, 4Cell® CHO Media, NutriStem® hPSC XF Medium, RoosterNourish™-MSC, Gibco Cell Culture Medium, DMEM, IMDM, etc. can be used depending on the bioprocess chosen. More preferably, a medium is used that provides optimal cell growth and proliferation rates, healthy physiology, morphology and / or appropriate gene expression.

[0084] Preferably, the second culture environment B is different from the first culture environment A in terms of the structural entities that establish the respective culture environments A, B. In these embodiments, the second culture environment B is provided by a second container separate from the first container 4. This allows the transfer of cells from the first container 4 to the second container. Preferably, when the proposed bioprocess installation 1 is put into operation, the second container does not contain the cell broth of the first container 4. The entities that establish the second culture environment B may be, for example, a second container such as a storage container or a second bioreactor.

[0085] As shown in Fig. 1, the clarification setup 5 preferably comprises a waste receptacle 16 designed as a waste container. Here, preferably, during the retraction run of the centrifuge 6, the liquid, preferably the solid particles in the discharged liquid, and more preferably the cells in the discharged liquid, are transferred under the control of the electronic process control device 2 to a second culture environment B or to a waste receptacle 21.

[0086] The chromatographic unit 8 comprises a stationary phase, which preferably consists of beads. These beads, which are preferably spherical, have a size of at least 250 μm in diameter, more preferably at least 350 μm in diameter.

[0087] According to another preferred embodiment, the stationary phase consists of at least one membrane, preferably a plurality of stacked membranes, or a monolith. According to this embodiment, at least one membrane and monolith has a plurality of pores. The pores have a pore size of more than 20 μm, preferably more than 40 μm.

[0088] Thus, in these embodiments of the stationary phase, particles with a size of up to 20 μm, preferably up to 40 μm, penetrate the pores of the stationary phase and possibly do not block the pores. Preferred materials for the stationary phase include agarose, polyamide, polysulfone, polyethersulfone (PES), polyvinylidene fluoride (PVDF), cellulose, cellulose acetate, silicone, polycarbonate, polyacrylonitrile (PAN), polyethylene (PE), polypropylene (PP), phenyl, etc.

[0089] It is particularly preferred to use these embodiments of the stationary phase of the chromatography unit 8 for affinity chromatography, in particular Protein A affinity chromatography, ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) or cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) or any other type of chromatography technique. Furthermore, these chromatography techniques may be operated in axial or radial flow and can additionally be combined with simulated moving bed chromatography to allow multiple parallel chromatography cycles. Thus, the chromatography unit comprises at least one chromatograph. At least one of these chromatographs comprises at least one chromatography column, preferably multiple chromatography columns.

[0090] In another particularly preferred embodiment, the bioprocessing unit 3 is designed for particle sizes between 10 μm and 100 μm, preferably between 15 μm and 80 μm, more preferably between 20 and 60 μm, so that solid particles, in particular cells, having a size up to 20 μm in diameter can be guided through the bioprocessing unit 3, preferably through the chromatography unit 8, without causing any blockage of the chromatography unit 8, in particular the chromatograph 10.

[0091] Preferably, at least one component of the bioprocessing unit 3, preferably at least one component of the vessel 4, the clarification unit 5, in particular the centrifuge 6 and / or the chromatography unit 8, more preferably all components of the bioprocessing unit 3, are designed as disposable components.

[0092] According to another independent teaching, a bioprocessing device 1 is claimed per se, which comprises an electronic process control device 2 and a bioprocessing unit 3. The bioprocessing unit 3 comprises at least one vessel 4, a clarification unit 5 with a centrifuge 6 and a chromatography unit 8 with a chromatograph 10. The cell broth obtained from the vessel 4 is first led through the clarification unit 5. The cell broth is then led through the chromatography unit 8. The passage of both units 5, 8 is carried out sequentially in a liquid flow 9. The clarification unit 5 with the centrifuge 6 is operated in a centrifugation cycle, which comprises centrifugation steps such as a filling step, a washing step and a draining step. On the other hand, the chromatography unit 8 with the chromatograph 10 is operated in a chromatography cycle, which comprises chromatographic steps such as an equilibration step, a filling step, a washing step, an elution step and a regeneration step.

[0093] What is important is that the particle reduction between the liquid flow section leaving the clarification unit 5 and the liquid flow section leaving the chromatography unit 8 is less than 10% in particle concentration, preferably less than 5%, and more preferably less than 2%, and that the execution of the steps assigned to the centrifugation cycle and the execution of the steps assigned to the chromatography cycle are at least partially synchronized with each other by the electronic process control device 2 in a synchronization routine 13 based on an assigned synchronization strategy 14.

[0094] The electronic process controller 2 is preferably designed to implement the proposed method by controlling the bioprocessing unit 3, the clarification unit 5 with the centrifuge 6 and / or the chromatography unit 8 with the chromatograph 10. The electronic process controller 2 may be realized as a central unit controlling all or at least most of the components of the bioprocessing installation 1. The electronic process controller 2 may also be realized in a distributed structure with multiple distributed units. In some embodiments, at least one electronic process controller 2 directs the opening and closing of one or more valves 17 (FIG. 1), the rotation speed of a rotor, either directly or via a motor, and / or the flow direction and / or velocity of fluids and / or particles from a vessel 4, such as a bioreactor.

[0095] Such an electronic process control device 2 comprises, for example, at least one digital control unit (DCU) and / or at least one multifermenter control system (MFCS) which itself comprises a local processor unit and a local data storage. The MFCS further provides a central process management system and dispatches requests to the digital control unit. Additionally or alternatively, such an electronic process control device 2 preferably comprises a computer and / or a server and / or a smartphone or the like. Here, preferably, the electronic process control device 2 has at least one microprocessor which is individually adjustable and / or programmable and / or capable of executing software. All the above explanations are fully applicable to this teaching.

[0096] According to another independent teaching, the electronic process control device of the bioprocessing installation 1 is claimed as such. Again, reference is made to all the above descriptions.

[0097] What is important is that the electronic process control device 2 is designed to carry out the proposed method by controlling a clarification unit 5, in particular a centrifuge 6, and / or a chromatography unit 8, in particular a chromatograph 10.

[0098] Preferably, the electronic process control device 2 comprises a data processing system 18 for the implementation of the above described methods, preferably comprising a local data storage and a local processor unit.

[0099] Finally, independent teachings relate to a computer program product for an electronic process control device 2 and a computer readable storage medium, on which the computer program product is preferably stored non-volatilely.

Claims

1. A method for manufacturing a bioproduct using a bioprocess facility (1), wherein the bioprocess facility (1) has an electronic process control device (2) and a bioprocess unit (3), and the bioprocess unit (3) has a container (4), a clarification unit (5) equipped with a centrifuge (6), and a chromatography unit (8) equipped with a chromatograph (10). The cell broth obtained from the container (4) is sequentially guided in a liquid stream (9) through the clarification unit (5) and the chromatography unit (8). The clarification unit (5) equipped with the centrifuge (6) is operated in a centrifugation cycle including centrifugation steps such as a filling step, a washing step, and a discharging step. The chromatography unit (8) equipped with the chromatograph (10) is operated in a chromatography cycle including chromatography steps such as an equilibration step, a filling step, a washing step, an elution step, and a regeneration step. In the method, the particle reduction between the liquid stream section exiting the clarification unit (5) and the liquid stream section exiting the chromatography unit (8) is less than 10% in particle concentration, and the execution of the steps assigned to the centrifugation cycle and the execution of the steps assigned to the chromatography cycle are at least partially synchronized with each other by a synchronization routine (13) based on an assigned synchronization strategy (14) by the electronic process control device (2).

2. The method according to claim 1, wherein the chromatography unit (8) receives the liquid stream section exiting the clarification unit (5) in an unfiltered state.

3. The method according to claim 1 or 2, wherein the chromatography unit (8) has a supply inlet directly connected to the outlet of the centrifuge (6).

4. The method according to claim 1, wherein the liquid stream (9) is at least temporarily continuous.

5. For at least one synchronization routine (13), the synchronization strategy (14) represents a temporal synchronization, and in accordance with the temporal synchronization, at least one centrifugation cycle remains in a predetermined temporal relationship with respect to at least one chromatography cycle, the method according to claim 1.

6. In accordance with the temporal synchronization, the cycles to be synchronized are started and / or ended simultaneously, or started and / or ended with a predetermined delay, the method according to claim 5.

7. In accordance with the temporal synchronization, one of the cycles to be synchronized is started depending on the start or end of each other cycle, the method according to claim 5 or 6.

8. For at least one synchronization routine (13), in addition to preferably representing a temporal synchronization, the synchronization strategy (14) additionally represents a liquid synchronization, and in accordance with the liquid synchronization, in at least one centrifugation cycle, liquid is transferred to the chromatography unit (8), the method according to claim 1.

9. For at least one synchronization routine (13), in accordance with the synchronization strategy (14), the filling step of the centrifugation cycle is synchronized with the filling step of the chromatography cycle, the method according to claim 1.

10. For at least one synchronization routine (13), in accordance with the synchronization strategy (14), the washing step of the centrifugation cycle is synchronized with the filling step and / or the washing step of the chromatography cycle, the method according to claim 1.

11. For at least one synchronization routine (13), according to the synchronization strategy (14), the discharge step of the centrifugation cycle is synchronized with any one of the equilibration step, the washing step, the elution step, and / or the regeneration step of the chromatography cycle, the method according to claim 1.

12. The cell broth is transferred from the first culture environment (A) established by the bioprocess unit (3) to the centrifuge (6), and in the reverse operation of the centrifuge (6), at least a part of the discharged cells is transferred into a separate second culture environment (B) different from the first culture environment (A) for subsequent cell culture and / or biological production, the method according to claim 1.

13. The chromatography unit (8) has a stationary phase, the stationary phase preferably consists of spherical beads, and the beads have a size with a diameter of at least 250 μm, more preferably a diameter of at least 350 μm, the method according to claim 1.

14. The chromatography unit (8) has a stationary phase, the stationary phase consists of at least one membrane, preferably a plurality of laminated membranes, or a monolith, and the stationary phase has a plurality of pores, the method according to claim 1.

15. The bioprocess unit (3) is designed to correspond to a particle size of 10 μm to 100 μm, preferably 20 to 40 μm, the method according to claim 1.

16. A bioprocess facility (1) comprising an electronic process control device (2) and a bioprocess unit (3), wherein the bioprocess unit (3) has a container (4), a clarification unit (5) comprising a centrifuge (6), and a chromatography unit (8) comprising a chromatograph (10). The cell broth obtained from the container (4) is sequentially guided in a liquid stream (9) through the clarification unit (5) and the chromatography unit (8). The clarification unit (5) comprising the centrifuge (6) is operated in a centrifugation cycle including centrifugation steps such as a filling step, a washing step, and a discharging step. The chromatography unit (8) comprising the chromatograph (10) is operated in a chromatography cycle including chromatography steps such as an equilibration step, a filling step, a washing step, an elution step, and a regeneration step. In the bioprocess facility (1), The particle reduction between the liquid stream section exiting the clarification unit and the liquid stream section exiting the chromatography unit (8) is less than 10% in terms of particle concentration. The execution of the steps assigned to the centrifugation cycle and the execution of the steps assigned to the chromatography cycle are at least partially synchronized with each other by a synchronization routine (13) based on an assigned synchronization strategy (14) by the electronic process control device (2). A bioprocess facility (1) is characterized in that.

17. The electronic process control device (2) is designed to implement the method according to claim 1 by controlling the clarification unit (5), in particular the centrifuge (6), and / or the chromatography unit (8), in particular the chromatograph (10). The bioprocess facility according to claim 16.

18. An electronic process control device for a bioprocess facility (1) according to claim 16, wherein the electronic process control device (2) is designed to implement the method according to claim 1 by controlling the clarification unit (5), in particular the centrifuge (6), and / or the chromatography unit (8), in particular the chromatograph (10). An electronic process control device characterized by this.

19. The electronic process control device (2) according to claim 18, wherein the electronic process control device (2) comprises a data processing system (18) for realizing the method according to claim 1.

20. A computer program product for an electronic process control device (2) according to claim 18 or 19.

21. A computer-readable storage medium, wherein the computer program product according to claim 20 is preferably stored non-volatilely.

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