Downstream bioprocessing process using batch tracking

Abstract

A bioprocessing process for separating a target species from a raw fluid mixture, the process comprising thep steps of (A) providing a raw fluid mixture comprising the target species; (B) purifying the raw fluid mixture yielding a fluid volume comprising the target species; (C) collecting the target species from the fluid volume; wherein step (B) comprises at least one series of sets of steps, wherein each series comprises at least two serially connected sets of steps, each set of steps comprising the steps of (a) processing an input fluid mixture comprising the target species in a bioprocessing unit to obtain an output fluid volume comprising the target species, wherein the bioprocessing unit comprises a unique unit id; (b) associating the output fluid volume with the bioprocessing unit yielding an association of output fluid volume and bioprocessing unit; (c) transferring the output fluid volume to a subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to step (C); wherein, if the set of steps is the first set of steps in the series of sets of steps, the input fluid mixture is the raw fluid mixture or wherein, if the set of steps is not the first set of steps in the series of sets of steps, the input fluid mixture is the output fluid volume of a previous set of steps directly preceding the set of steps, and wherein in step (b) each association of output fluid volume and bioprocessing unit is stored.

Description

[0001] Downstream Bioprocessing Process Using Batch Tracking

[0002] Technical Field

[0003] The present invention is directed towards downstream bioprocessing processes, in particular towards downstream bioprocessing processes having improved quality control.

[0004] Background

[0005] Downstream bioprocessing refers to the recovery and the purification of biosynthetic products from natural sources such as animal tissue, plant tissue, or fermentation broth, thereby recycling salvageable components from these resources.

[0006] Downstream bioprocessing is an essential step in the manufacture of pharmaceuticals such as antibiotics, hormones (e.g., insulin and human growth hormone), antibodies (e.g., infliximab and abciximab), vaccines, antibodies and enzymes used in diagnostics, industrial enzymes, natural fragrance, and flavour compounds.

[0007] It is widely accepted to categorize downstream bioprocessing into four stages, which are applied to transform a product from its natural or post-synthetic state as a component of a tissue, cell, or fermentation broth into the pure form of the product, thereby owing to high purity standards depending on the further intended use of the product, e.g., in medicine or analytics. These four stages are generally recognized as removal of insoluble (sometimes also regarded as midstream bioprocessing), product isolation, product polishing, and product formulating.

[0008] Removal of insoluble (midstream bioprocessing) involves the capture of the product as a solute in a particulate-free liquid. Hence, e.g., cells, cell debris, or other particulate matter has to be separated from a liquid comprising the product. Typically, the removal of insoluble is achieved by filtration, centrifugation, sedimentation, precipitation, flocculation, electro-precipitation, and / or gravity settling. Additional operations such as grinding, homogenization, or leaching, required to recover products from solid sources such as plant and animal tissues, are usually included in this group.

[0009] Product isolation comprises the removal of components having physical and chemical properties varying considerably from the properties of the product. In bioprocessing, this mostly includes the removal of water. As such, product isolation reduces the volume of the material to be processed and further concentrates the product. Typically, product isolation is achieved by solvent extraction, adsorption, ultrafiltration, and / or precipitation. Product polishing is dedicated to separate the products from components having very close physical and chemical properties. Hence, product purification involves expensive steps, which require sensitive and sophisticated equipment. This stage constitutes a major part of the entire downstream bioprocessing process. Typical steps used in product purification are chromatography steps (e.g., affinity, size exclusion, hydrophobic interaction, reversed phase, ion-exchange) as well as crystallization and fractional precipitation.

[0010] Product formulating describes final processing steps typically yielding packaging of the product in a stable and easily form. Crystallization, desiccation, lyophilization and spray drying are typical unit operations. Depending on the product and its intended use, product formulation may also include operations to sterilize the product.

[0011] Certain operations combine two or more of the described stages. For example, expanded bed adsorption accomplishes removal of insolubles and product isolation in a single step. As another example, affinity chromatography can isolate and polish in a single step.

[0012] In industrial application it is desired to achieve downstream bioprocessing in an efficient and cost-reduced manner. Therefore, processes have been developed, which connected operation steps as described above, which transport the product in a liquid phase to facilitate the transportation from one step to the other. Furthermore, it the outputs of several preceding operational steps can be collected as a combined input for a subsequent step. This is due to the concentration effect usually achieved by each step. In the beginning of a bioprocess, the volume liquid per product is very high, wherein during purification the concentration of the product is significantly enhanced. Hence, at least part of bioprocesses is typically set up in tree-like structures, whereas at the beginning of the downstream bioprocessing process many operational units are present in the first step, whereas the number of operational units usually gets fewer and fewer with each step downstream in the process.

[0013] As a result, in the final step only a few outputs are present, into which the outputs of a variety of multiple operational units have been processed. Such a setup constitutes high demands in view of product quality control. Quality control, however, is a general demand in downstream bioprocessing, as the purity of the product is usually an important and desired property.

[0014] Furthermore, it is a general desire when running a batchwise, semi-continuous process to know where each batch is or has been at which time. Apart from quality improvement, such knowledge can be used, e.g., when designing and setting up bioprocessing processes. Furthermore, it can also be used to ensure traceability for medical products or in general. Summary of the Invention

[0015] Usually, quality of the product in each of the outputs is controlled by analytical units. Furthermore, also analytical units can be placed in between any of the preceding steps of the downstream bioprocessing process. However, depending on the operational units and the analytics used, there can be a timely gap in between the information retrieved from the analytical unit and the transport of the product batch to the next subsequent operational unit. Such a timely gap, however, leads to the problem that it is not known anymore where a potentially faulty product batch has been transported in the meantime. As a result, the product of the whole product tree has to be removed as waste leading to a significant loss of material and product.

[0016] To solve this problem, the downstream bioprocessing process could be set up in a way that each operational unit waits for the result of the analytical unit before the product is transported to the next step. However, this either results in a significant slowdown of the process or the requirement of a significantly increased number of operational units to achieve the same production speed.

[0017] The timely gap in analytical units not only affects the decision to discard certain faulty product batches, but also the ability to optimize the process e.g., by optimizing parameters of preceding steps based on the outcome of the analytical units.

[0018] Finally, there is generally no fine-grained control and trackability of the product batches in the whole downstream bioprocessing process.

[0019] Thus, there is the need of a downstream bioprocessing process, which allows for precise and timely accurate removal of product batches, which have been identified as fault by an analytical unit, even if this analytical unit needs a significant time period for delivering the result such as an off-line analytical unit. Furthermore, there is the need for a downstream bioprocessing process, which allows for fine-grained disposal of faulty product batches, thereby allowing for saving valuable resources, product, and costs, and eliminating or at least reducing downtime.

[0020] It has now surprisingly been found that above-mentioned objects can be achieved by a bioprocessing process for separating a target species from a raw fluid mixture, the process comprising the steps of:

[0021] (A) providing a raw fluid mixture comprising the target species;

[0022] (B) purifying the raw fluid mixture yielding a fluid volume comprising the target species;

[0023] (C) collecting the target species from the fluid volume; wherein step (B) comprises at least one series of sets of steps, wherein each series comprises at least two serially connected sets of steps, each set of steps comprising the steps of: (a) processing an input fluid mixture comprising the target species in a bioprocessing unit to obtain an output fluid volume comprising the target species;

[0024] (b) associating the output fluid volume with the bioprocessing unit yielding an association of output fluid volume and bioprocessing unit;

[0025] (c) transferring the output fluid volume to a subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to step (C); wherein, if the set of steps is the first set of steps in the series of sets of steps, the input fluid mixture is the raw fluid mixture or wherein, if the set of steps is not the first set of steps in the series of sets of steps, the input fluid mixture is the output fluid volume of a previous set of steps directly preceding the set of steps, and wherein in step (b) each association of output fluid volume and bioprocessing unit is stored.

[0026] In various aspects, the present invention has the advantage that at any time and in any state of the process, a product batch can be identified and in particular located with respect of the bioprocessing units used in the downstream bioprocessing process. Thereby, if a certain product batch is known to be faulty, it can be removed before it reaches the final output thereby contaminating other product batches having been produced in parallel bioprocessing units, as well as subsequent or preceding steps of the same bioprocessing unit.

[0027] Brief Description of the Drawings

[0028] Figure 1 is a schematic visualization of the steps of a preferred embodiment of the bioprocessing process of the present invention including a normal flow filtration step (NFF), a capture chromatography step, a conditioning step, a polishing step, a tangential flow filtration step (TFF), and an analysis step.

[0029] Figure 2 is a schematic visualization of the steps of a preferred embodiment of the bioprocessing process of the present invention including a capture chromatography step, a conditioning step, a polishing step, and a tangential flow filtration step (TFF), wherein chromatograms of two pathways of fluid volumes are exemplified (A and B). Visualizations may be possible in both directions upstream to downstream or downstream to upstream.

[0030] Figure 3 is a schematic visualization of the steps of the preferred embodiment of the bioprocessing process of the present invention according to Figure 1 including failed states 1 to 5. Figure 4 is a schematic visualization of the setup of the bioprocessing process used in the first example of example C.

[0031] Figure 5 is a schematic visualization of the steps of the preferred embodiment of the bioprocessing process of the present invention according to Figure 4 (first example of C).

[0032] Figure 6 is a schematic visualization of the steps of the preferred embodiment of the bioprocessing process of the present invention according to Figure 4, but with different transfers (second example of C).

[0033] Figure 7 is a schematic visualization of the setup of the bioprocessing process used in the second example of example D.

[0034] Definitions

[0035] The term “fluid volume" as used herein denotes a volume comprising a fluid and preferably the target species. Preferably, the fluid is a liquid. More preferably, the fluid is a liquid comprising, preferably consisting of, a solvent. Even more preferably, the solvent is selected from water-based buffer solutions, such as phosphate, acetate, citrate, or tris(hydroxymethyl)aminomethane, and mixtures thereof with other solvents such as ethanol. The solvents can comprise additives such as polymers, e.g., PEG and / or dextran, salts, e.g., ammonium sulphate, sodium chloride etc., detergents, and / or stabilizers. Some affinity stationary phases (membranes or beads) may require more complex mixtures. For instance, Immobilized Metal Chelate Affinity Chromatography (IMAC) requires the addition of an organic compound such as imidazole.

[0036] The term “target species" as used herein denotes the product to be isolated. Usually, the target species is a substance naturally occurring in animal or plant tissue, is produced by naturally occurring microorganisms or enzymes, and / or is produced by modified, preferably genetically modified, microorganisms or enzymes. As such, the target species can be, but is not limited to, antibodies, proteins, viruses, organic substances, DNA molecules, RNA molecules, shorter nucleotides molecules, exosomes, cells (such as used in cell therapy), or specific target molecules.

[0037] The term “raw mixture" as used herein denotes a mixture of various contaminant, solvents, and at least one target species. The mixture can be a natural product, such as animal or plant tissues, which already has been brought in contact with at least one solvent, or a mixture from a bioreactor. Preferably, the raw mixture is a mixture from a bioreactor comprising microorganisms, such as yeasts, bacteria, and / or fungi, cells, viruses, tissue, target species, and / or solvents. The term “flow-through mode" as used herein denotes a chromatography method, in which impurities in a raw mixture are bound to the chromatography matrix, while the target species is not bound, thereby separating the target species in the dilute.

[0038] The term “bind / elute mode" as used herein denotes a chromatography method, in which the target species in a raw mixture is bound to the chromatography matrix, while the impurities are not bound. The impurities are washed from the column with the dilute and the column is flushed with an elute, which removes the target species from the chromatography column, thereby separating the target species in the elute.

[0039] The term “bioprocessing unit" as used herein denotes a unit, which receives a fluid volume comprising a target species and usually contaminants and which outputs a fluid volume comprising the target species and usually less contaminants. Preferably, the bioprocessing unit is selected from the list consisting of chromatographic devices, including columns and membranes, filters, centrifuges, extraction devices, and two- phase separation devices.

[0040] The term “off-line analysis" as used herein denotes an analysis step, wherein the sample is taken out of the bioprocessing unit or a transportation line in sterile conditions and analysed in a laboratory after physical pre-treatments (e.g., filtration and dilution). Preferably, the preparation and handling require clear Standard Operating Procedures (SOPs) as well as skilled personnel. Together with the complexity involved in manual handling, the major disadvantage of off-line measurement is the time delay, which results in lower measurement frequency. Due to these issues off-line measurements are usually not considered true PAT (Process Analytical Technology) unless there are no other measurement possibilities (e.g., HPLC for product titter or mass spectroscopy for product quality). Off-line laboratory measurements are commonly used to monitor and validate the accuracy of the in-line / on-line process analysers.

[0041] The term “at-line analysis" as used herein denotes an analysis step, wherein the sample is removed from the bioprocessing unit or a transportation line and is analysed in close proximity to the production process, either manually or by using automated sampling devices. Similar to off-line measurement, sterile conditions must be maintained for accurate results. At-line measurement is most common for parameters which cannot be measured accurately in-line or on-line. Advantages of at-line measurement include shortened time delay (relative to off-line), and the possibility for automated control.

[0042] The term "on-line analysis" as used herein denotes an analysis step, wherein the sample is diverted from the bioprocessing unit or a transportation line by a branching and may be returned to the bioprocessing process after analysis. The sample is automatically measured in the by-pass by process sensors. The advantages of this method can be found in the simple sterilization and the straightforward access to the sample in stationary conditions. The implementation of such a solution requires a specifically designed or modified bioprocessing process, in particular bioprocessing unit and / or transportation line.

[0043] The term “in-line analysis" as used herein denotes an analysis step, wherein the measurement occurs directly in the bioprocessing unit or the transportation line with a process sensor. Preferably, the generated measurements are sent in real-time to the control system. Process parameters such as pH, ORP (redox potential), dissolved oxygen, dissolved CO2, temperature, and / or conductivity are commonly measured by in-line analysis. Moreover, also measurements allowing for detection of specific molecules can be measured by in-line analysis, such as LIV / VIS absobance / transmittance, fluorescence emission, and refractive index.

[0044] The term “unique id" as used herein denotes an identification information, which is unique in the address space used for the object to be identified. Hence, it is assured that the id is never used for two objects in the same address space.

[0045] The term “association" as used herein denotes a link between two pieces of information, preferably a unique link. In case of the bioprocessing process of the present invention an association for example can provide a link between a bioprocessing unit and an output fluid volume by linking the unique unit id with the unique output fluid volume id. Hence, e.g., pointers, data tables, or arrays are preferred as associations. Preferably, the arrays are multidimensional arrays. The preferred associations are dedicated sets of parameters, which have the advantage that further parameters can be added to said association such as start and end date and time as well as an error state value.

[0046] As used in this specification and in the appended claims, the singular forms of “a“ and “an" also include the respective plurals unless the context clearly dictates otherwise. In the context of the present invention, the terms “about" and “approximately" denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±8 %, more preferably ±5 %, even more preferably ±2 %. It is to be understood that the term “comprising" and “encompassing" is not limiting. For the purposes of the present invention the term “consisting of‘ is considered to be a preferred embodiment of the term “comprising of‘. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only. Furthermore, the terms “first" , “second", “third" or “(a)“, “(b)“, “(c)“, “(d)" etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. In case the terms “first", “second", “third11or “(a)“, “(b)“, “(c)“, “(d)“, “i“, “K“ etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e. the steps may be carried out simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in the application as set forth herein above or below. It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0047] As used herein the term “does not comprise", “does not contain", or “free of’ means in the context that the composition of the present invention is free of a specific compound or group of compounds, which may be combined under a collective term, that the composition does not comprise said compound or group of compounds in an amount of more than 0.8 % by weight, based on the total weight of the composition. Furthermore, it is preferred that the composition according to the present invention does not comprise said compounds or group of compounds in an amount of more than 0.5 % by weight, preferably the composition does not comprise said compounds or group of compounds at all.

[0048] When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100% (± 1 % due to rounding).

[0049] Detailed Description

[0050] In one aspect, the present invention is related to a downstream bioprocessing process being able to identify the product batches produced therein. Usually, downstream bioprocessing processes are not continuous processes. This is due to the character of the process, which is defined by the connection of several bioprocessing units or steps, which themselves usually are designed to deliver the product batch-wise. Hence, usually, the downstream bioprocessing processes are set up in a semi-continuous manner. As such the product is distributed all over the process in batches, which can be found in the bioprocessing units themselves, i.e., being processed, in transport lines in between the bioprocessing units, i.e., being transported, or in hold-up tanks in between bioprocessing units, i.e., waiting for further processing. To be able to identify each of these product batches, the present invention relates to a bioprocessing process for separating a target species from a raw fluid mixture, the process comprising the steps of:

[0051] (A) providing a raw fluid mixture comprising the target species;

[0052] (B) purifying the raw fluid mixture yielding a fluid volume comprising the target species;

[0053] (C) collecting the target species from the fluid volume; wherein step (B) comprises at least one series of sets of steps, wherein each series comprises at least two serially connected sets of steps, each set of steps comprising the steps of:

[0054] (a) processing an input fluid mixture comprising the target species in a bioprocessing unit to obtain an output fluid volume comprising the target species;

[0055] (b) associating the output fluid volume with the bioprocessing unit yielding an association of output fluid volume and bioprocessing unit, wherein the bioprocessing unit comprises a unique unit id;

[0056] (c) transferring the output fluid volume to a subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to step (C); wherein, if the set of steps is the first set of steps in the series of sets of steps, the input fluid mixture is the raw fluid mixture or wherein, if the set of steps is not the first set of steps in the series of sets of steps, the input fluid mixture is the output fluid volume of a previous set of steps directly preceding the set of steps, and wherein in step (b) each association of output fluid volume and bioprocessing unit is stored.

[0057] With the help of the association between the fluid volume and the bioprocessing unit, it is assured that at any time of the process the fluid volume can be located. Hence, once it is known that said volume is faulty, it can be removed by transporting it into waste instead of transporting it to the subsequent steps and / or set of steps and finally to step (C). Thereby, transporting into waste could also be understood in a way that the faulty volume remains in a container in the bioprocessing unit, wherein the container is emptied at the next opportunity for cleaning the bioprocessing unit. The association of output fluid volume and bioprocessing unit can be achieved in any way so that after the association has been stored it can be uniquely retrieved and identified. Hence, preferably, the association is made between two unique identification information (id), one unique id for the bioprocessing unit and one unique id for the output fluid volume either by a direct or indirect link. In a first preferred embodiment of the present invention, in the bioprocessing process step (b) comprises the step of associating the output fluid volume and the input mixture yielding an association of output fluid volume and input mixture, wherein each association between output fluid volume and input mixture is stored.

[0058] With this information, the pathway of output fluid volumes can be retrieved by the information stored. For example, if the information of a last output fluid volume is known and it should be investigated, where in the process this output fluid volume has been travelling, it simply has to be followed the link to the input mixture of the last set of steps, which itself is an output fluid volume of at least one previous set of steps. As this previous output fluid volume also corresponds to another data stored, again, its preceding output fluid volume id can be determined and so forth. This approach yields the total pathway of the last output fluid volume in the bioprocessing process. Hence, for example, each processing unit can be located, by which a faulty fluid volume has been processed. This can be helpful to identify the root cause for the failure in the fluid volume. It can be further helpful to track down a faulty bioprocessing unit if necessary. Furthermore, it helps finding all other fluid volumes, with which the faulty batch has been in contact or united to ensure removal of all fluid volumes, which might be affected by the faulty fluid volume.

[0059] Even more preferably, in the bioprocessing process according to first preferred embodiment of the present invention, step (b) further comprises the steps of

[0060] (ba.2) creating a set of parameters associated with said output fluid volume, wherein the set of parameters comprises at least a unique fluid volume id and a unit id, wherein the unique fluid volume id is created, and the unique unit id of the bioprocessing unit is added to the set of parameters;

[0061] (ba.3) adding a list of preceding fluid volume ids to the set of parameters, if it is not already present, and adding at last one unique preceding fluid volume id to the list of preceding volume ids, wherein the at least one unique preceding fluid volume id comprises the unique fluid volume id of each output fluid volume of each of the previous set of steps directly preceding the set of steps, or wherein, if the set of steps is the first set of steps, the unique preceding fluid volume id is the unique fluid volume id of the raw fluid mixture, wherein the unique fluid volume id of the raw fluid mixture is created.

[0062] Hence, in the first preferred embodiment of the present invention, each output fluid volume receives its own unique output fluid volume id. It also receives the respective unique unit id thereby creating a clear association between the output fluid volume and the bioprocessing unit it has been processed on. As the data also comprises a list of all previous output fluid volume ids, the pathway through the bioprocessing process can be recreated.

[0063] Most preferably, in the bioprocessing process according to first preferred embodiment of the present invention, step (b) further comprises the steps of

[0064] (ba.4) adding a list of subsequent fluid volume ids to each of at least one previous set of parameters of each of the at least one previous set of steps directly preceding the set of steps, if not already present, and

[0065] (ba.5) adding the output fluid volume id to each list of subsequent fluid volume ids of each of the at least one previous set of parameters of each of the at least one previous set of steps directly preceding the set of steps.

[0066] This has the advantage that the recreation of the pathway works in both directions, i.e., upstream and downstream. Thus, the pathway can be recreated in any direction starting from any step within the bioprocessing process. This saves time and computing resources.

[0067] In a second preferred embodiment of the present invention, in step (b) of the bioprocessing process according to the present invention each association of output fluid volume and bioprocessing unit is stored in the order of its timely occurrence. This also assures that the pathway of a fluid volume along bioprocessing units can be recreated. Hence, for example, each processing unit can be located, by which a faulty fluid volume has been processed. This can be helpful to identify the root cause for the failure in the fluid volume. It can be further helpful to track down a faulty bioprocessing unit if necessary. Furthermore, it helps finding all other fluid volumes, with which the faulty batch has been in contact or united to ensure removal of all fluid volumes, which might be affected by the faulty fluid volume.

[0068] Hence, preferably, in the bioprocessing process according to the second preferred embodiment of the present invention, step (b) comprises the step of

[0069] (bb.1) if the set of steps is the first set of steps in the series of sets of steps, creating a set of parameters associated with said output fluid volume, wherein the set of parameters comprises at least a unique fluid volume id and a unit id collection, wherein the unique fluid id is created, and the unique unit id of the bioprocessing unit is added to one end, preferably the bottom, of the unit id collection; or

[0070] (bb.2) if the set of steps is not the first set of steps in the series of sets of steps, adding the unique unit id of the bioprocessing unit to one end, preferably the bottom, of the unit id collection of the set of parameters.

[0071] Hence, the bioprocessing process of the present invention creates the unique fluid id on the fly upon first processing of such fluid volume. In case the bioprocessing unit is not able to provide a unique unit id, step (bb.2) could also include the step of creating a unique unit id. From the unique ids and their association in the common set of parameters, the location of the fluid volume can be retrieved.

[0072] Even more preferably, in the bioprocessing process according to the present invention step (b) comprises a step of associating a start time and date and an end time and date with the association of output fluid volume and bioprocessing unit. In this embodiment it is not necessary to store each association of output fluid volume and bioprocessing unit in the order of its timely occurrence. Rather, the timely occurrence can be computed from the start and end date and time. Furthermore, the residence time of the fluid volume in the bioprocessing unit is also recorded. The residence time can be further compared to other residence times of other fluid volumes in the bioprocessing unit. Furthermore, the residence time can be compared to residence times of other fluid volumes in other, parallel, bioprocessing units. Thereby, as faulty bioprocessing unit can be identified. Preferably, the start time and date are recorded when the fluid input mixture enters the bioprocessing unit, and the end time and date is recorded when the output fluid mixture has left the bioprocessing unit.

[0073] In another preferred embodiment step (b) of the bioprocessing process of the present invention comprises a step of associating a unit status value with the association of output fluid volume and bioprocessing unit. Thus, if an error state is detected for a bioprocessing unit while the fluid volume has been processed, such error state can be associated with the association of output fluid volume and bioprocessing unit. Hence, in case a path of the fluid volume is recreated, the error state information can be directly retrieved from the storage. Preferably, the step (b) further comprises the step of evaluating a total output fluid volume status value from the unit status values associated with said output fluid volume. More preferably, the step of evaluating a total output fluid volume status value returns an error if at least one of the status values associated with said output fluid volume represents an error. Thus, if an error occurs anywhere along the pathway of a fluid volume in the bioprocessing process of the present invention, the fluid volume is marked erroneous or faulty. This allows for certain actions to be triggered even if, e.g., the last bioprocessing unit by which the fluid volume has been processed, does not output an error state. Preferably, such action could be the removal of the faulty fluid volume. Hence, preferably, if the step of evaluating a total output fluid volume status value returns an error, the output fluid volume is discarded, and step (c) is not carried out.

[0074] Hence, preferably, in the bioprocessing process according to the present invention, the step of associating a start time and date and an end time and date with the association of output fluid volume comprises a step of adding a start time and date and an end time and date associated with said unique unit id to the set of parameters. Likewise, preferably, in the bioprocessing process according to the present invention, the step of associating a status value with the association of output fluid volume and bioprocessing unit comprises a step of adding a status value associated with said unique unit id to the set of parameters. Hence, this assures that the relation between the bioprocessing unit, the fluid volume, the residence times of the fluid volume in the bioprocessing unit, and potential error states of the bioprocessing unit and resulting faulty fluid volume can be retrieved from one place in the data storage.

[0075] Even more preferably, in the bioprocessing process according to the present invention step (b) comprises a step of associating detector data of the output fluid volume with the association of output fluid volume and bioprocessing unit. Preferably, the detector data has been received by a detector selected from the list consisting of a Ultraviolet light absorption (UV) detector, a visible light absorption (VIS) detector, a photo diode array (PDA) detector, a refractive-index detector, an evaporative light scattering detector, a multi-angle light scattering detector, a mass spectrometer, a conductivity detector, a fluorescence detector, a chemiluminescence detector, an optical rotation detector, and an electrochemical detector. Preferably, the detector data comprises at least one signal of the target species. The association with the detector data allows further quality control, as for each fluid volume processed at any time in the bioprocessing process of the present invention, the time-dependent occurrence of the target species and / or impurities in the fluid volume can be controlled during and after the process. Furthermore, the detector data can be used to determine an error state of the bioprocessing unit, e.g., by comparison of the detector data to stored detector data.

[0076] Preferably the collected data of each fluid volume are delivered until the end of the bioprocessing process. This ensures quality control from the beginning to the end. Hence, preferably, in the bioprocessing process according to the present invention, the step (c) comprises the step of:

[0077] (c.1) Transferring the set of parameters in parallel to step (c).

[0078] In a preferred embodiment of the present invention, the bioprocessing units are in communication with a central control unit and the association of output fluid volume and bioprocessing unit is stored on said control unit or wherein the bioprocessing units are in communication with each other, and the association of output fluid volume and bioprocessing unit is stored on the bioprocessing unit and is transferred in step (c) in parallel to the output fluid volume.

[0079] The central control unit could be any device being able to process and store data. Thereby, it is not necessary that the device is able to store the data persistently. However, in view of reliability and quality control it is preferable that the central control unit can store the data persistently. The data may be stored in a file such a simple file format. Preferably, the data is stored in a database, such as a Structured Query Language database. As such, the central control unit could be a computer, preferably a computer with a persistent storage such a hard disk.

[0080] However, a central control unit is not necessarily needed. As the bioprocessing devices nowadays are usually equipped with a processing unit, these units could also be used instead of a central processing unit. In such an embodiment the data must be transport from one bioprocessing unit to the subsequent bioprocessing unit such that the data travels alongside with the fluid volume along pathway of the fluid volume.

[0081] Hence, preferably, in the bioprocessing process according to the present invention, the step (c) comprises, if the bioprocessing unit is in communication with a central control unit, the step of:

[0082] (c.2) Transferring the set of parameters to the central control unit; or, if the bioprocessing units are in communication with each other, the step of:

[0083] (c.2) Transferring the set of parameters to the bioprocessing unit of the subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to a collecting unit of step (C).

[0084] Preferably, the bioprocessing process is set up in that at least one consecutive step has lower numbers of bioprocessing units than the step before. Reason is that the fluid volume can be reduced from step to step to achieve a higher concentration of the target species. Hence, preferably, in the bioprocessing process according to the present invention, in at least one series of sets of steps a set of steps receives the output fluid volumes of at least two previous sets of steps directly preceding said set of steps. In this way, two or more sets of steps deliver their output fluid volumes to one subsequent set of steps. Thereby the total volume of the fluid volume is increased and a potential reduction of the volumes in the preceding sets of steps is compensated.

[0085] Occasionally, it can be desired to set up a process, in which a set of steps is followed by more than one sets of steps. This might be the case, if, e.g., a spare bioprocessing unit should be provided, which could be used in case one of the other parallel bioprocessing units enters an error state. However, it could also be used to speed up a step, which normally is significantly slower than the preceding step. Hence, preferably, in the bioprocessing process according to the present invention, in at least one series of sets of steps the output fluid volume of a set of steps is split into partial output fluid volumes, wherein each of the partial output fluid volumes are transferred to one of at least two subsequent sets of steps of the series of sets of steps directly following the set of steps, wherein the number of partial output fluid volumes equals the number of the at least two subsequent sets of steps, and wherein to each of the at least two subsequent sets of steps only one partial fluid volume is transferred. Usually, the parallel setup as described for the preferred embodiment above affords that the outputs of the parallel sets of steps are again combined and transported to a single subsequent sets of step. Hence, preferably, in the bioprocessing process according to the present invention, the at least two previous sets of steps are identical to the at least two subsequent sets of steps.

[0086] To determine a faulty fluid volume, the volume is usually analysed. Hence, preferably, in the bioprocessing process according to the present invention, at least one of the sets of steps comprises an analysis step of subjecting at least a part of the output fluid volume to an analysis step prior to step (c). Preferably, the analysis step comprises, preferably consists of, a step selected from an in-line analysis step, an on-line analysis step, an at-line analysis step, and / or an off-line analysis step. More preferably, the analysis step comprises, preferably consists of, a step selected from an in-line analysis step, an on-line analysis step, and / or an at-line analysis step. Even more preferably, the analysis step comprises, preferably consists of, a step selected from an in-line analysis step, and / or an on-line analysis step. Most preferably, the analysis step comprises, preferably consists of, an in-line analysis step.

[0087] In a preferred embodiment of the bioprocessing process of the present invention, upon detecting an error in the analysis step, the status value associated with the bioprocessing unit of the processing step preceding the analysis step in the parameter set is set to an error. As such, the fluid volume is marked as faulty and preferably is discarded in one of the following steps. This ensures that as little as possible product is discarded from the bioprocessing process and assuring maximum purity achievable with the bioprocessing process. Even more preferably, the bioprocessing unit, which has a status value set to an error, can be identified, and restored to resume production of this bioprocessing unit. Restoration can be achieved by replacing the faulty bioprocessing unit and by identifying the root cause of the error and removal thereof.

[0088] Preferably, each step a) of the bioprocessing process of the present invention could be any process step related to the four stages of downstream bioprocessing, i.e. , removal of insolubles, product isolation, product purification, and product polishing. More preferably, each step a) of the bioprocess of the present invention is selected from the list consisting of a clarification step, a capturing step, a conditioning step, a polishing step, and a sterilization step. Preferably, the clarification step is selected from the list consisting of coagulation, flocculation, centrifugation, sedimentation, and filtration, more preferably from the list consisting of depth filtration, normal flow filtration (NFF), continuous centrifugation, and tangential flow filtration (TFF). Preferably, the capturing step is a chromatography step in bind / elute mode selected from the list consisting of affinity chromatography, cation exchange chromatography, and anion exchange chromatography. In a preferred embodiment of the present invention, the capturing steps can be carried out as multi column chromatography (MCC) or periodic countercurrent chromatography (PCC). The conditioning step is preferably selected from the list consisting of a pH adjustment step, a conductivity adjustment step, and a viral inactivation step. Finally, the polishing step preferably is a chromatography or (membrane) filtering step in flow through mode, whereas the chromatography method is selected in view of the target species to be separated. However, preferably, the polishing step is selected from the list consisting of affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, and anion exchange chromatography.

[0089] In an especially preferred embodiment of the present invention, step B) of the bioprocessing process comprises at least five sets of steps, whereas step a) of the first set of steps is a clarification step, preferably a normal flow filtration step, step a) of the second set of steps is a capturing step, preferably an affinity chromatography step in bind / elute mode, step a) of the third set of steps is a conditioning step, preferably a pH adjustment step, step a) of the fourth set of steps is a polishing step, preferably an affinity chromatography step in flow-through mode, and step a) of the fifth set of steps is a tangential flow filtration step. Preferably, the output fluid volumes of multiple set of steps according to the first set of steps are received by each set of steps according to the second set of steps, most preferably the output fluid volumes of six set of steps according to the first set of steps are received by each set of steps according to the second set of steps. Likewise, preferably, the output fluid volumes of multiple set of steps according to the fourth set of steps are received by each set of steps according to the fifth set of steps, most preferably the output fluid volumes of eight set of steps according to the fourth set of steps are received by each set of steps according to the fifth set of steps. Furthermore, preferably, the bioprocessing process according to the especially preferred embodiment further comprises at least one set of steps comprising an analysis step of subjecting at least a part of the output fluid volume to an analysis step prior to step (c). Preferably, all set of steps of the bioprocessing process according to the especially preferred embodiment comprise an analysis step of subjecting at least a part of the output fluid volume to an analysis step prior to step (c).

[0090] In a second preferred embodiment of the present invention, the target species is at least one monoclonal antibody, and the impurities include host cell proteins, DNA, and other contaminants. Preferably, step B) of the bioprocessing process of the second preferred embodiment comprises six sets of steps, whereas step a) of the first set of steps is a clarification step, preferably selected from the list consisting of depth filtration, normal flow filtration, and continuous centrifugation, step a) of the second set of steps is an affinity capturing step, preferably selected from the list consisting of affinity chromatography in bind / elute mode and affinity membrane filtration, step a) of the third set of steps is a conditioning step, preferably a pH adjustment step, a conductivity adjustment step and a virus inactivation step, step a) of the fourth set of steps is a polishing step, preferably multimodal chromatography step in flow-through mode or an anion exchange membrane filtration step, step a) of the fifth set of steps is a tangential flow filtration step, and step a) of the sixth set of steps is a sterile filtration step.

[0091] In a third preferred embodiment of the present invention, the target species is an adeno associated virus (AAV). Preferably, step B) of the bioprocessing process of the second preferred embodiment comprises five sets of steps, whereas step a) of the first set of steps is a clarification step, preferably selected from the list consisting of depth filtration, normal flow filtration, and continuous centrifugation, step a) of the second set of steps is an affinity capturing step, preferably selected from the list consisting of affinity chromatography in bind / elute mode and cation exchange chromatography in bind / elute mode, step a) of the third set of steps is a dilution step, step a) of the fourth set of steps is a polishing step, preferably an anion exchange chromatography step in flow-through mode, and step a) of the fifth set of steps is a sterile filtration step.

[0092] In a first especially preferred embodiment, step B) of the bioprocessing process of the present invention comprise at least six steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0093] Generally, the first especially preferred embodiment of the bioprocessing process of the present invention is directed towards monoclonal antibody (mAb) purification and, thus, to a process for removing host cell proteins (HCP), DNA, and other contaminants while maintaining the integrity of the product. It is important to effectively remove aggregates, which can be very toxic and reduce mAb therapeutic efficacy.

[0094] Hence, the first step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step.

[0095] Furthermore, the second step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for affinity capturing, such as an affinity chromatography step (e.g., Cytiva MabSelect Sure or MabSelect PrismA resins or any other Protein A-based resin) or an affinity membrane step (e.g., Sartorius Sartobind Rapid A affinity membrane or any other Protein A-based membrane). Moreover, the third step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for conditioning, such as a viral inactivation step, or a pH / conductivity adjustment step.

[0096] The fourth step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for polishing, such as a multimodal chromatography step (e.g., Cytiva Capto adhere or Capto adhere ImpRes), an anion exchanger chromatography (AIEX) step (e.g., Cytiva Capto Q resin), or an anion exchanger membrane step (e.g., Pall Mustang Q).

[0097] Furthermore, the fifth step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0098] Finally, the sixth step of the bioprocessing process according to the first especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0099] In the bioprocessing process, the affinity capturing step and the polishing step are the two most important steps. The first provides a concentration of mAb molecules, while the second is run in flow-through mode (mAbs pass the column without binding, only impurities, such as aggregates, HCP or DNA, bind).

[0100] In a second especially preferred embodiment the step B) comprises at least eight steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0101] Generally, the second especially preferred embodiment of the bioprocessing process of the present invention is directed towards adeno associated virus (AAV) purification.

[0102] The first step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for cell lysis.

[0103] Hence, the second step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step.

[0104] Furthermore, the third step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0105] Moreover, the fourth step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for affinity capturing, such as an affinity chromatography step (e.g., Cytiva Capto AVB resin, Thermofisher Poros Capture Select AAVX resin) or a cation exchanger chromatography (CIEX) step.

[0106] The fifth step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for dilution.

[0107] The sixth step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for AIEX polishing, such as an anion exchanger chromatography (AIEX) step (e.g., Cytiva Capto Q resin).

[0108] Furthermore, the seventh step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0109] Finally, the eighth step of the bioprocessing process according to the second especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0110] In the bioprocessing process, the affinity capturing step and the AIEX polishing step are the two most important steps. The first provides a concentration of AAV, while the second provides an effective separation of full and empty adeno-associated virus capsids (viral particles with no viral genome).

[0111] In a third especially preferred embodiment the step B) comprises at least seven steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0112] Generally, the third especially preferred embodiment of the bioprocessing process of the present invention is directed towards adeno virus (AdV) purification. Adeno viruses are used as viral vectors for gene therapy or vaccines and as oncolytic viruses. Because of this wide application range, adeno virus production volumes can vary significantly. Hence, the interest in scalable platforms for industrial adenovirus production is increasing.

[0113] The first step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for cell lysis. This step preferably is suitable for a process, in which the adeno viruses are released from the infected cells (upstream culture) and cell DNA is chopped down in smaller fragments with benzonase.

[0114] Hence, the second step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step. Furthermore, the third step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0115] The fourth step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for capturing, such as an anion exchanger chromatography (AIEX) step in bind / elute mode (e.g., Cytiva Q Sepharose XL or Cytiva Capto Q ImpRes).

[0116] Moreover, the fifth step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for polishing, such as a size exclusion chromatography step (e.g., Cytiva Sepharose 4 Fast Flow) or a multimodal chromatography step (e.g., Cytiva Capto Core 700).

[0117] Furthermore, the sixth step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0118] Finally, the seventh step of the bioprocessing process according to the third especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0119] In the bioprocessing process, the capturing step and the polishing step are the two most important steps. In the first step, negatively charged adeno viruses bind to the AIEX resin, while in the second step they flow through the chromatographic resin.

[0120] In a fourth especially preferred embodiment the step B) comprises at least seven steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0121] Generally, the fourth especially preferred embodiment of the bioprocessing process of the present invention is directed towards lentvirus (LV) purification. Lentviruses (LV) are enveloped RNA viruses belonging to the retroviridae family with a particle size of -80-100 nm. LV are one of the most efficient gene transfer vectors and integrate the RNA into the host cell DNA. LV is commonly used for Chimeric Antigen Receptor (CAR) T cell therapy to successfully treat cancer. Purification of lentiviral vector is very challenging due to low stability of this enveloped virus that is sensitive to low pH, high salt, temperature, and shear forces for example.

[0122] The first step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for harvest nuclease. Hence, the second step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step.

[0123] Furthermore, the third step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0124] The fourth step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for + / - capturing, such as an anion exchanger chromatography (AIEX) step in bind / elute mode (e.g., Cytiva Capto DEAE resin, Pall Mustang Q membrane).

[0125] Moreover, the fifth step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for polishing, such as a multimodal chromatography step (e.g., Cytiva Capto Core 700 resin).

[0126] Furthermore, the sixth step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0127] Finally, the seventh step of the bioprocessing process according to the fourth especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0128] In the bioprocessing process, the capturing step and the polishing step are the two most important steps. In the first step, a concentration of LV and an efficient purification of the viral particles is provided, whereas the second step, which is operated in flow- through mode, the viruses do not bind to the chromatography resin, while only the impurities bind.

[0129] In a fifth especially preferred embodiment the step B) comprises at least five steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0130] Generally, the fifth especially preferred embodiment of the bioprocessing process of the present invention is directed towards exosome purification. Exosomes are a class of cell-derived extracellular vesicles of endosomal origin and are typically 30-150 nm in diameter. Enveloped by a lipid bilayer, exosomes are released into the extracellular environment containing a complex cargo of contents derived from the original cell, including proteins, lipids, mRNA, miRNA and DNA. These naturally-equipped nanovesicles can be therapeutically targeted or engineered as drug delivery systems. The first step of the bioprocessing process according to the fifth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0131] The second step of the bioprocessing process according to the fifth especially preferred embodiment of the present invention is a step for chromatographic purification, such as a multimodal chromatography step (e.g., Cytiva Capto Core 700 resin) or a size exclusion chromatography step (e.g., Cytiva Sephacryl S-400 HR).

[0132] Moreover, the third step of the bioprocessing process according to the fifth especially preferred embodiment of the present invention is a step for chromatographic purification, such as an ion exchanger chromatography step (e.g., Cytiva Capto Q or Cytiva Fibro Q or Pall Mustang Q).

[0133] Furthermore, the fourth step of the bioprocessing process according to the fifth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0134] Finally, the fifth step of the bioprocessing process according to the fifth especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0135] In the bioprocessing process, the two chromatographic purification steps are the two most important steps. In the first step, the exosomes flow through the beads, whereas smaller particles / impurities enter the pores in the beads. Furthermore, preferably, the ion exchange chromatography step is an anion exchanger working in bind / elute mode.

[0136] In a sixth especially preferred embodiment the step B) comprises at least nine steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0137] Generally, the sixth especially preferred embodiment of the bioprocessing process of the present invention is directed towards plasmid DNA (pDNA) purification. A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning and are in principle used for the cell and gene therapy industry. For instance, they are used to produce mRNA or AAVs used in gene therapy. The first step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for cell lysis and flocculation. In this step destruction of bacteria (E-Coli) occurs to extract the plasmids and flocculation of cell debris.

[0138] Hence, the second step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step.

[0139] Furthermore, the third step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0140] The fourth step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for normal flow filtration.

[0141] Moreover, the fifth step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for chromatographic purification, such as a size exclusion chromatography step (e.g., Cytiva Sepharose 6 FF).

[0142] The sixth step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for chromatographic purification (e.g., Cytiva Plasmid Select Xtra or Capto Plasmid Select).

[0143] The seventh step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for AIEX polishing, such as an anion exchanger chromatography (AIEX) step (e.g., Cytiva Source 30 Q).

[0144] Furthermore, the eighth step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0145] Finally, the ninth step of the bioprocessing process according to the sixth especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0146] In the bioprocessing process, three chromatographic steps are involved: size exclusion chromatography, removal of open circular (OC) pDNA, and anion exchanger chromatography. In the size exclusion chromatography, the large pDNA flows through the beads, whereas smaller RNA enters the pores in the beads. In the OC pDNA removal step, the OC and SC (supercoiled conformation) pDNA conformations are eluted selectively, whereas the AIEX polishing is used to fine purify the SC pDNA in a bind-elute mode. In a seventh especially preferred embodiment the step B) comprises at least eight steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0147] Generally, the seventh especially preferred embodiment of the bioprocessing process of the present invention is an alternative approach directed towards plasmid DNA (pDNA) purification.

[0148] The first step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for cell lysis and flocculation. In this step destruction of bacteria (E-Coli) occurs to extract the plasmids and flocculation of cell debris.

[0149] Hence, the second step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for clarification, such as a depth filtration step, a normal flow filtration step, or a continuous centrifugation step.

[0150] Furthermore, the third step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0151] The fourth step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for normal flow filtration.

[0152] Moreover, the fifth step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for chromatographic purification, such as an anion exchange membrane step in bind / elute mode (e.g., Pall Mustang Q XT140).

[0153] The sixth step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for chromatographic purification (e.g., Cytiva Plasmid Select Xtra or Capto Plasmid Select).

[0154] Furthermore, the seventh step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0155] Finally, the eighth step of the bioprocessing process according to the seventh especially preferred embodiment of the present invention is a step for sterile filtration, such as a 0.2 pm filtration step with sterile grade filters.

[0156] In the bioprocessing process, only two chromatographic steps are involved: Al EX chromatography and removal of OC pDNA. The anion exchanger chromatography is run using a charged membrane instead of a chromatographic resin, and the normal the classical OC pDNA removal step as in the previous process.

[0157] In an eighth especially preferred embodiment, the step B) comprises at least six steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0158] Generally, the eighth especially preferred embodiment of the bioprocessing process of the present invention is directed towards messenger RNA (mRNA) purification. Messenger RNAs are single-stranded nucleic acids transcribed from DNA. When used as a preventative vaccine or a therapeutic drug, mRNA is typically delivered to the cell’s cytoplasm where it directs production of protein-based antigens. By mimicking the actions of natural mRNAs, therapeutical mRNAs use cells as natural “bioreactors” to produce clinically relevant proteins, thus avoiding challenges associated with some protein-based therapeutics.

[0159] The upstream step is a step for in vitro transcription, i.e., a stirred tank bioreactor. Thereby, mRNA is produced by incubating DNA template with an RNA polymerase - usually the T7-RNA-Polymerase - and nucleotides (NTPs) in a cell-free in vitro transcription (IVT) process.

[0160] The first step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0161] Furthermore, the second step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for affinity capturing, such as an affinity chromatography step (e.g., a highly specific chromatographic resin or membrane based on Oligo-DT ligands).

[0162] The third step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for tangential flow filtration.

[0163] The fourth step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for polishing, such as a multimodal chromatography step (e.g., Cytiva Capto Core 700 resin) or a hydrophobic interaction chromatography (HIC) step (e.g., Cytiva Butyl Sepharose 4 Fast Flow).

[0164] Furthermore, the fifth step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for tangential flow filtration. Finally, the sixth step of the bioprocessing process according to the eighth especially preferred embodiment of the present invention is a step for liquid nanoparticle encapsulation of the purified mRNA.

[0165] In a ninth especially preferred embodiment, the step B) comprises at least five steps, which are described in the following, preferably serially connected in the order as provided in the following. It should be further understood that this embodiment is not limited to the actual number of steps. Hence, some of the steps as provided herein might be present more than once in the bioprocessing process.

[0166] Generally, the ninth especially preferred embodiment of the bioprocessing process of the present invention is directed towards oligo synthesis. DNA oligonucleotides can be manufactured via chemical synthesis using a fully automated oligonucleotide synthesizer like the Cytiva AKTA oligosynt. However, the resulting oligo molecule needs to go through a purification procedure to achieve product quality as required for pharmaceutical / therapy use.

[0167] Hence, the upstream bioprocessing step is an oligonucleotide synthesis.

[0168] The first step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for cleavage and protection. Preferably, the first step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is suitable for ester hydrolysis of the linker and simultaneous removal of the product from the solid support, both of which are carried out by treatment with concentrated aqueous ammonia.

[0169] Furthermore, the second step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for normal flow filtration (NFF) and tangential flow filtration (TFF) to remove solid matter and adopt concentration and buffer.

[0170] The third step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for polishing, such as an anion exchange chromatography (AIEX) step (e.g., using a Cytiva Capto Q resin).

[0171] The fourth step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for desalting and concentration, i.e., either by size exclusion chromatography (for small scale) (e.g., Sephacryl S-300 HR) or by tangential flow filtration (for large scall).

[0172] The fourth step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for polishing, such as a by multimodal chromatography bioprocessing unit (e.g., Cytiva Capto Core 700 resin) or by hydrophobic interaction chromatography (HIC) (e.g., Cytiva Butyl Sepharose 4 Fast Flow).

[0173] Finally, the fifth step of the bioprocessing process according to the ninth especially preferred embodiment of the present invention is a step for sterile filtration, such as 0.2 pm filtration with sterile grade filters.

[0174] In an alternative embodiment of the ninth especially preferred embodiment of the present invention, the third step is preceded by two more steps, wherein the first of these steps is for chromatography purification (i.e., HIC or RPC), and the second of these steps is suitable for Detritylation (acid pH).

[0175] The key element in this alternative embodiment of the ninth especially preferred embodiment of the present invention is that the hydrophobic protective trityl group (DMTr) is retained after the upstream step and used as a handle for hydrophobic interaction chromatography (HIC) (e.g., Cytiva Phenil Sepharose 6 FF) or reversephase chromatography (RPC) (e.g., Cytiva Source 30 RPC) purification. The trityl group is then easily removed by lowing pH using a strong acid solution. Then the polishing step is achieved using a regular anion exchange chromatography resin.

[0176] Examples

[0177] Figure 1 depicts a schematic visualization of a preferred embodiment of the bioprocessing process of the present invention. This preferred bioprocessing process comprises five sets of steps, each comprising a step a), wherein the first set of steps implements a normal flow filtration (NFF) as step a), the second set of steps implements a capturing step as step a), the third set of steps implements a conditioning step as step a), the fourth set of steps implements a polishing step as step a), and the fifth set of steps implements a tangential flow filtration (TFF) step as step a). Finally, in the surge tank connecting the first and the second sets of steps, an at-line analysis step is implemented. It can be seen that the whole process is set up in a tree-like fashion. Hence, the second set of steps implementing the capturing step as step a) receives the output fluid volumes of up to six preceding sets of steps implementing a normal flow filtration step as step a). Likewise, the fifth set of steps implementing a tangential flow filtration step as step a) receives eight output fluid volumes from all preceding fourth sets of steps implementing a polishing step as step a). During the conditioning step usually no significant change in volume is achieve, in particular not a decrease in volume. Hence, the output fluid volume of the third sets of steps implementing a condition step as step a) are large enough to feed the subsequent sets of steps implementing a polishing step as step a). Likewise, as the sets of steps implementing a capturing step as step a) usually capture more than one fluid volume at once, the output volume of them is also large enough to feed the subsequent sets of steps implementing a conditioning step as step a).

[0178] Figure 2 shows a section of the process of Figure 1 , i.e., selecting only the sets of steps implementing a capturing step, a conditioning step, a polishing step, and a tangential flow filtration step as steps a). For example, pathways of a fluid volume, the information stored for each bioprocessing unit in said pathway has been shown (i.e., A and B).

[0179] Example A

[0180] In Example A, a fluid volume is followed from the capturing step via the condition step, the polishing step to the tangential flow filtration step. The resulting information (unique id of the bioprocessing unit encoded as a colour, start and end time indicated by the start and end of the chromatograms per each colour, and the detector data as the values depicted by each of the chromatograms per each colour. By this information, the flow of the fluid volume in the process can be controlled at any time the fluid volume has been or is processed.

[0181] Example B

[0182] In example B, the information stored in view of one of the sets of steps implementing a tangential flow filtration as step a) has been shown. Likewise, the resulting information comprises the unique id of the bioprocessing unit encoded as order and colour, the start and end time indicated by the start and end of the chromatograms per each order and colour, and the detector data as the values depicted by each of the chromatograms per each order and colour. From this information, it can be retrieved that each fluid volume, which has entered the set of steps implementing a tangential flow filtration as step a) has been successfully processed, i.e., is not a faulty fluid volume.

[0183] Figure 3 shows a schematic visualisation according to Figure 1 indicating several scenarios, in which the current implementation provides advantages. Item @ indicates a deviation in the loading time of the capture column. As the fluid volume is still in the bioprocessing unit while the error is recognized by the system, the fluid volume is routed to an escape route to a waste vessel still from the same bio processing unit. Item @ indicates a deviation in the elution time of the capture column. As the elute is normally directly routed to the next set of steps implementing a conditioning step as step a), the system activates the routing to an escape route in the bioprocessing unit of the set of steps implementing a conditioning step as step a). In both cases, the faulty output fluid volumes of items @ and @ are removed before entering the set of steps implementing a tangential flow filtration as step a). Thereby, the whole process remains operational and the output volume of the set of steps implementing a tangential flow filtration as step a) does not have to be discarded, but still can be used as a regular product batch with ensured quality. Item @ indicates a deviation in an analysis result. As the analysis step is a fast analysis step, the fluid volume still can be routed to an escape route in the same bioprocessing unit. The situation is different in item @, where the analysis is slower, and the fluid volume has been already transferred to the next set of steps implementing a conditioning step as step a). In this case the system routes the faulty fluid volume from the next bioprocessing unit to an escape route. Again, the whole process remains operational and the output volume of the set of steps implementing a tangential flow filtration as step a) does not have to be discarded, but still can be used as a regular product batch with ensured quality.

[0184] Item @ indicates a deviation in the set of steps implementing a TFF step as step a) or even a manual call for an escape route. In this case, the fluid volume is simply routed to an escape route and the process remains fully operational at ensured quality of the product.

[0185] Example C

[0186] Considering Figure 4, which shows a system consisting of 4 Akta bioprocessing units (System 1-4) connected by three transfer systems, i.e., tubes in this case (ts1-3). Each bioprocessing unit is connected to a computer, on which a master program runs, which controls client programs running on the same computer. These client programs control the UniCorn API of the Akta bioprocessing units. The master further controls a database for storing and reading data.

[0187] Whenever a bioprocessing system completes one process cycle and releases an output volume, a new volume is registered, and a database entry is therefore created. Most of the attributes are empty or have a default value at time of creation and are filled later. The database entry that is created looks like this: wherein:

[0188] “_id” is the id of the volume constructed from two attributes “client_id” and

[0189] “cycle” separated by an underscore;

[0190] “client_id” is the id of the bioprocessing unit;

[0191] “cycle” is the cycle number for the bioprocessing unit, starts at 1 and is incremented by 1 for each cycle of the bioprocessing unit. For example, 1_2 is the _id for the second cycle on bioprocessing unit 1 ;

[0192] “next_id” is a list of _id's of any volumes originating from the current volume;

[0193] “sample” is a relation between a sample and the volume, if any; “source_id” is a list of _id's of the volumes the current batch is originating from;

[0194] “start” is the start time of the cycle that the volume corresponds to;

[0195] “status” is the status of the bioprocessing unit / volume. It is set to accepted by default and then changed to failed after a failed sample analysis or if manually set to failed during the run.

[0196] “loadStart” is the start time for loading of the bioprocessing unit.

[0197] “loadStop” is the end time for loading of the bioprocessing unit.

[0198] “elutionStart” is the start time of delivery by the bioprocessing unit.

[0199] “elutionStop” is the end time of delivery by the bioprocessing unit.

[0200] “cycleEnd” is the end time of the cycle that the volume corresponds to.

[0201] If the bioprocessing unit starts a new cycle, a “new cycle” message is sent from the client to the master. A new entry for the new volume is created in the master database. The master fills the "start” attribute in the database entry with the time the message was received. Now the bioprocessing unit does all steps necessary for being ready to receive a sample, such as equilibration.

[0202] When, e.g., equilibration is complete, the client sends a “ready to receive” message to the master and pauses while waiting for the go-ahead. When the master sends the go- ahead to the client it also fills out the time of the attribute “loadStart” in the database entry for the current volume on that client. Furthermore, the master also enters the _id's of the volumes used to feed the bioprocessing unit in the “source_id” list in the database entry for the volume. As the list of batches is copied to the database entry for the current volume, the master also goes to the database entries of each of these volumes in the list and add the current volume to the “next_id” list for each of those volumes. Then, the bioprocessing unit is allowed to load the sample.

[0203] When all instructions for loading have been completed by the client, the client sends a “loading complete” message to the master. The master receives the message and enters the time to the “loadStop” attribute in the database entry for the current volume on that client. Washing is performed on the bioprocessing unit by the client.

[0204] When washing is complete, the client sends a “ready to deliver” message to the master and pauses while waiting for the go-ahead. When the master sends the go-ahead to the client it enters the time as the attribute “elutionStart” in the database entry for the current volume on that client. Elution (delivery) is performed on the bioprocessing unit by the client. When all instructions for delivery have been completed by the client it sends a “delivery complete” message to the master. The master receives the message and enters the time as the “elutionStop” attribute in the database entry for the current volume on that client. When the next cycle starts with a “new cycle” message the master enters the time as the “cycleEnd” attribute in the database.

[0205] Figure 5 shows the database entries after a run has completed for a short run of the process of Figure 4.

[0206] The coloured line for each system visualizes the time when a batch is inside a system and grey lines visualizes which batches are connected through the chain built from “next_id” and “source_id” in the database entries.

[0207] The start and end points for the colored line is taken from the database entry attributes “loadStart” and “elutionEnd”. The batch is then delivered by a transfer marked by a blue circle. The data displayed by Figure 5 is shown in the following representation:

[0208] { "_id" : "1 1", "client_id" : "1" "cycle" : 1, "next_id" : [ "2_1" ], "sample" : [ ], "source_id" : [ ], "start" : 24.11, "status" : "accept", "loadstart" : 34.63, "loadstop" : 45.1, "elutionStart" : 55.62,

[0209] "elutionStop" : 66.12, "cycleEnd" : 76.61 }

[0210] { "_id" : "4_1", "client_id" : "4", "cycle" : 1, "next_id" : [ ], "sample" : [ ], "source_id" : { "3_1" : 1 }, "start" : 24.11, "status" : "accept", "loadstart" : 129.1, "loadstop" : 139.6, "elukionStart" :

[0211] 150.11, "elutionStop" : 160.61, "cycleEnd" : 171.1 }

[0212] { "_id" : "2_1", "client_id" : 1, "next_id" : [ "3_1" ], "sample" : [ ], "source_id" : start" : 24.62, "status" : accept", "loadstart" : 66.12, 76.64, "elutionStart" : 87.12, "elutionStop" : 97.62, "cycleEnd" : 108.11 }

[0213] { "_id" : "3_1", "client_id" : "3", "cycle" : 1, "next_id" : [ "4_1" "sample" : [ ], "source_id" : { "2_1" : 1 }, "start" : 24.62, "status" : "accept", "loadstart" : 97.62, "loadstop" : 198.11, "elutionStart" : 118.63, "elutionStop" : 129.1, "cycleEnd" : 139.6 }

[0214] { "_id" : 25200 "client_id" : "cycle" : 2, "next_id" : [ "2_2" ], "sample" : [ ], "source_id" : [ ], "start" : 76.61, "status" : "accept", "loadstart" : 87.12, "loadstop" : 97.62, "elutionStart" :

[0215] 108.11, "elutionStop" : 118.63 }

[0216] { "_id" : "2 2", "client_id" : "2", "cycle" : 2, "next_id" : [23_2"], "sample" : [ ], "source_id" : { "1_2" : 1 }, "start" : 108.11, "status" : "accept", "loadstart" : 118.63, "loadstop" : 129.1, "elutionStart" : 139.6, "elutionStop" : 150.11 }

[0217] { "_id" : "3_2", "client_id" : "3", "cycle" : 2, "next_id" : [ "4_2" ], "sample" : [ ], "source_id" : { "2_2" : 1 }, "start" : 139.6, "status" : "accept", "loadstart" : 150.11, "loadstop" : 160.61, "elutionStart" :

[0218] 171.1, "elutionStop" : 181.61 }

[0219] { "_id" : "4_2", "client_id" : "4", "cycle" : 2, "next_id" : [ "4_2" ], "sample" : [ ], "source_id" : { "3_2" : 1 }, "start" : 171.1, "status" : "accept", "loadstart" : 181.61, "loadstop" : 192.1, "elutionStart" : 202.61, "elutionStop" : 213.13 }

[0220] Thereby, the first volume produced in System 1 (left blue bar) corresponds to the first entry marked bold. The second marked bold entry corresponds to the second volume produced in System 2 (i.e., right yellow bar).

[0221] Figure 6 shows a more complex example. In this example a 2:1 transfer exists between System 1 and System 2, and a 1:2 transfer between System 3 and System 4. This can clearly be seen in the graph where two batches are pooled before they are loaded into system 2 and one batch from system 3 becomes two batches in system 4. We can also see the same in the database. For example, both batch 1_1 and 1_2 has batch 2_1 as their “next_id” and batch 2_1 has two “source_id”, 1_1 and 1_2. The data is shown below.

[0222] { "_id" : "1 1" , "client_id" : "1", "cycle" : 1, "next_id" : [ "2_1" ], sample : [ ], "source_id" : [ ], "start" : 53.62, "status" : "accept", "loadstart" : 64.12, "loadstop" : 74.62, "elutionStart" : 85.13, elutionStop 179.62, "cycleEnd" : 106.11 }

[0223] { "_id" : "2_1", "client_id" : "2", "next_id" : [ "3_1" ], "sample" [ ], "source_id" { "1 1" ; "start" : 53.62, "status" :

[0224] "accept", "loadstart" : 148.1, "loadstop" : 158.61, "elutionStart" :

[0225] 169.1, "elutionStop" : 179.62, "cycleEnd" 190.13 }

[0226] { "_id" : "3_1", "client_id" : "3", "cycle" : 1, "next_id" [ "4_1", "4_2"), "sample" : [ ], "source_id" : { "2_1" : 1}, "start" : 53.62, "status" : "accept", "loadstart" : 179.62, "loadstop" : 190.13, "elutionStart" : 200.6, "elutionStop" : 211.12, "cycleEnd" : 221.62 }

[0227] { "_id" : "4_1", "client_id" : "4", "cycle" : 1, "next_id" : [ ], "sample" : [ ], "source_id" { "3_1" : 1}, "start" : 53.62, "status" : "accept", "loadstart" : 211.12, "loadstop" : 221.62, "elutionStart" :

[0228] 232.1, "elutionStop" : 242.61, "cycleEnd" : 253.12 }

[0229] { "_id" : "1_2", "client_id" : "1" "cycle" : 2, "next_id" : [ "2_1" ], "sample" : [ ], "source_id" : [ ], "start" : 106.11, "status" : "accept", "loadstart" : 116.61, "loadstop" : 127.11, "elutionStart" : 137.61, "elutionStop" : 148.1, "cycleEnd" : 158.61 } { "_id" : "1_3", "client_id" : "3", "cycle" : 3 "next_id" : [ "2_2" ], "sample" : [ ], "source_id" : [ ], "start" : 158.61, "status" : "accept", "loadstart" : 169.1, "loadstop" : 179.62, "elutionStart" : 190.13, "elutionStop" : 200.6, "cycleEnd" : 211.12 }

[0230] { "_id" : "2_2", "client_id" : "2", "cycle" : 2, "next_id" : [ "3_2" ], "sample [ ], "source_id" : { "1_3" : 1, "1_4" : 1}, "start" : 190.13, "status" : "accept", "loadstart" : 253.12, "loadstop" : 263.62, "elutionStart" : 274.12, "elutionStop" : 284.63 }

[0231] { "_id" : "1_4", "client_id": "1", "cycle" : 4, "next_id" : [ "2_2" ], "sample" : [ ], "source_id" : [ ], "start" : 211.12, "status" : "accept", "loadstart" : 221.62, "loadstop" : 232.11, "elutionStart" : 242.61, "elutionStop" : 253.12 }

[0232] { "_id" : "3_2", "client_id" : "3", "cycle" : 2, "next_id" : [ "4_3", "4_4" ], "sample" : [ ], "source_id" : { "2_2" : 1 }, "start" : 221.62, "status" : "accept", "loadstart" : 284.63, "loadstop" : 295.11, "elutionStart" : 305.6, "elutionStop" : 316.11 }

[0233] { "_id" : "4_2", "client_id" : "4", "cycle" : 2, "next_id" : [ ], "sample" : [ ], "source_id" : { "3_1" : 1 }, "start" : 253.12, "status" : "accept", "loadstart" : 263.62, "loadstop" : 274.12, "elutionStart" : 284.63, "elutionStop" : 295.11, "cycleEnd" : 305.6 }

[0234] { "_id" : "4_3", "client_id" : "4", "cycle" : 3, "next_id" : [ ], "sample" : [ ], "source_id" : { "3_2" : 1 }, "start" : 305.6, "status" : "accept", "loadstart" : 316.11, "loadstop" : 326.63, "elutionStart" : 337.1, "elutionStop" : 347.62, "cycleEnd" : 358.11 }

[0235] { "_id" : "4_4", "client_id" : "4", "cycle" : 4, "next_id" : [ ], "sample" : [ ], "source_id" : { "3_2" : 1 }, "start" : 358.11, "status" : "accept", "loadstart" : 368.62, "loadstop" : 379.12, "elutionStart" : 389.6, "elutionStop" : 400.11 }

[0236] Example D

[0237] A database entry for sampling (i.e., branching) differs from a database for regular volumes. The data for it looks like indicated in the following:

[0238] { "_id" : "4_sl", "client_id" : "4", "cycle" : 1, "next_id" : [ "4_1" ], "sample_tag" : "4_sl", "source_client" : "4", "source_id" : [ "1 1" ], " source_type" : "transfer", "start" : 65.6, "status" : "accept", " subbatch_id" : "4_sl", "samplestart" : 67.12 } First of all, the _id of the volume is generated such as done in example C, however, a “s” is added to the string to indicate that it is a sample batch (i.e., raw mixture). The attribute “source_type” shows whether the sample is from a “transfer” or directly from a valve port on a bioprocessing unit. During a transfer, more than one volume could be part of the sample.

[0239] The setup for Example D is depicted in Figure 7, which shows three systems with 1 : 1 transfers in between. On the first transfer a sampling / analysis system is connected, which samples from the transfer. Systems 1 , 2, and 3 work just as shown in Example C. System 4 differs from the rest of the system. It does not use the usual “ready to receive” / ”loading complete” messages in its instruction list. Instead, when it is ready to receive a sample, it sends a different message to the master to generate a sample tag which creates the database entry above for 4_s1. At this point client 4 has sent both a “new cycle” message and a “generate sample tag” message. This means that there is one entry for volume 4_1 and one entry for 4_s1 in the database. The sample volume works as a bridge between the source volume in t1 and volume 4_1 as can be seen in the entry for 4_s1 , where source id is 1_1 and next id is 4_1. This also means that volume 4_1 in the database would have the “sample” attribute filled out with volume 4_s1 to be able to link an analysis result back to that volume later down the line when the analysis is complete. After this initial difference from the regular transfer flow, System 4 works exactly as any other system with “ready to deliver” / ’’delivery complete” messages.

[0240] Sampling is normally carried out to analyze the sample. Hence, after the analysis, the result thereof is sent to the master using the message “analysis complete”. The master checks, which batch that system is currently processing and which sample the currently processed batch is linked to through the “sample” attribute. Then, the result is stored and linked to this batch. The master also compares the result to predefined thresholds. If the result is outside of the thresholds, the sample batch is marked as failed and all batches from the “source” of the sample batch will also be marked as failed. Subsequently, all batches originating from these source batches will then also be marked as failed. This is done by following the “next_id” attribute to the end.

Claims

Claims:

1. A bioprocessing process for separating a target species from a raw fluid mixture, the process comprising the steps of:(A) providing a raw fluid mixture comprising the target species;(B) purifying the raw fluid mixture yielding a fluid volume comprising the target species;(C) collecting the target species from the fluid volume; wherein step (B) comprises at least one series of sets of steps, wherein each series comprises at least two serially connected sets of steps, each set of steps comprising the steps of:(a) processing an input fluid mixture comprising the target species in a bioprocessing unit to obtain an output fluid volume comprising the target species, wherein the bioprocessing unit comprises a unique unit id;(b) associating the output fluid volume with the bioprocessing unit yielding an association of output fluid volume and bioprocessing unit;(c) transferring the output fluid volume to at least one subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to step (C); wherein, if the set of steps is the first set of steps in the series of sets of steps, the input fluid mixture is the raw fluid mixture or wherein, if the set of steps is not the first set of steps in the series of sets of steps, the input fluid mixture is the output fluid volume of at least one previous set of steps directly preceding the set of steps, wherein in step (b) each association of output fluid volume and bioprocessing unit is stored.

2. The bioprocessing process according to claim 1 , wherein step (b) comprises the step of(ba.1) associating the output fluid volume and the input fluid mixture yielding an association of output fluid volume and input fluid mixture, wherein each association between output fluid volume and input fluid mixture is stored.

3. The bioprocessing process according to claim 2, wherein step (b) further comprises the steps of(ba.2) creating a set of parameters associated with said output fluid volume, wherein the set of parameters comprises at least a unique fluid volume idand a unit id, wherein the unique fluid volume id is created, and the unique unit id of the bioprocessing unit is added to the set of parameters;(ba.3) adding a list of preceding fluid volume ids to the set of parameters, if it is not already present, and adding at last one unique preceding fluid volume id to the list of preceding volume ids, wherein the at least one unique preceding fluid volume id comprises the unique fluid volume id of each output fluid volume of each of the previous set of steps directly preceding the set of steps, or wherein, if the set of steps is the first set of steps, the unique preceding fluid volume id is the unique fluid volume id of the raw fluid mixture, wherein the unique fluid volume id of the raw fluid mixture is created.

4. The bioprocessing process according to claim 3, wherein step (b) further comprises the steps of(ba.4) adding a list of subsequent fluid volume ids to each of at least one previous set of parameters of each of the at least one previous set of steps directly preceding the set of steps, if not already present, and(ba.5) adding the output fluid volume id to each list of subsequent fluid volume ids of each of the at least one previous set of parameters of each of the at least one previous set of steps directly preceding the set of steps.

5. The bioprocessing process according to claim 1 , wherein in step (b) each association of output fluid volume and bioprocessing unit is stored in the order of its timely occurrence.

6. The bioprocessing process according to claim 5, wherein step (b) comprises the steps of(bb.1) if the set of steps is the first set of steps in the series of sets of steps, creating a set of parameters associated with said output fluid volume, wherein the set of parameters comprises at least a unique fluid volume id and a unit id collection, wherein the unique fluid volume id is created, and the unique unit id of the bioprocessing unit is added to one end, preferably the bottom, of the unit id collection; or(bb.2) if the set of steps is not the first set of steps in the series of sets of steps, adding the unique unit id of the bioprocessing unit to one end, preferably the bottom, of the unit id collection of the set of parameters.

7. The bioprocessing process according to any of the preceding claims, wherein step (b) comprises a step of associating a unit status value with the association of output fluid volume and bioprocessing unit.

8. The bioprocessing process according to any of the preceding claims, wherein the step of associating a status value with the association of output fluid volume and bioprocessing unit comprises a step of adding a status value associated with said unique unit id to the set of parameters.

9. The bioprocessing process according to any of the preceding claims, wherein the step (b) further comprises the step of evaluating a total output fluid volume status value from the unit status values associated with said output fluid volume.

10. The bioprocessing process according to claim 9, wherein the step of evaluating a total output fluid volume status value returns an error if at least one of the status values associated with said output fluid volume represents an error.11 . The bioprocessing process according to claim 10, wherein the output fluid volume is discarded, and step (c) is not carried out, if the total output fluid volume status value represents an error.

12. The bioprocessing process according to any of the preceding claims, wherein in at least one series of sets of steps a set of steps receives the output fluid volumes of at least two previous sets of steps directly preceding said set of steps.

13. The bioprocessing process according to any of the preceding claims, wherein in at least one series of sets of steps the output fluid volume of a set of steps is split into partial output fluid volumes, wherein each of the partial output fluid volumes are transferred to one of at least two subsequent sets of steps of the series of sets of steps directly following the set of steps, wherein the number of partial output fluid volumes equals the number of the at least two subsequent sets of steps, and wherein to each of the at least two subsequent sets of steps only one partial fluid volume is transferred.

14. The bioprocessing process according to claims 12 and 13, wherein the at least two previous sets of steps are identical to the at least two subsequent sets of steps.

15. The bioprocessing process according to any of the preceding claims, wherein at least one of the sets of steps comprises an analysis step of subjecting at least a part of the output fluid volume to an analysis step prior to step (c).

16. The bioprocessing process according to claim 15, wherein the analysis step comprises, preferably consists of, a step selected from an inline analysis step, an online analysis step, or an external analysis step.

17. The bioprocessing according to claims 15 or 16, wherein upon detecting an error in the analysis step, the status value associated with the bioprocessing unit of the processing step preceding the analysis step in the parameter set is set to an error.

18. The bioprocessing process according any of the preceding claims, wherein step (b) comprises a step of associating a start time and date and an end time and date with the association of output fluid volume and bioprocessing unit.

19. The bioprocessing process according to claim 18, wherein the start time and date is recorded when the fluid input mixture enters the bioprocessing unit, and the end time and date is recorded when the output fluid mixture has left the bioprocessing unit.

20. The bioprocessing process according to any of the preceding claims 18 or 19, wherein the step of associating a start time and date and an end time and date with the association of output fluid volume comprises a step of adding a start time and date and an end time and date associated with said unique unit id to the set of parameters.

21. The bioprocessing process according to any of the preceding claims, wherein the bioprocessing units are in communication with a central control unit and the association of output fluid volume and bioprocessing unit is stored on said control unit or wherein the bioprocessing units are in communication with each other, and the association of output fluid volume and bioprocessing unit is stored on the bioprocessing unit and is transferred in step (c) in parallel to the output fluid volume.

22. The bioprocessing process according to claim 21 , wherein the step (c) comprises, if the bioprocessing unit is in communication with a central control unit, the step of:(c.1) transferring the set of parameters to the central control unit; or, if the bioprocessing units are in communication with each other, the step of:(c.1) transferring the set of parameters to the bioprocessing unit of the subsequent set of steps of the series of sets of steps directly following the set of steps or, if the set of steps is the last set of steps in the series of sets of steps, to a collecting unit of step (C).

Images