Continuous preparation of biological substances with process traceability

Abstract

The present invention relates to a method and a system for the continuous preparation of a biological substance with process traceability. The methods and systems involve using a bioreactor to perform multiple cell culture and harvest cycles to produce batches of biological material, wherein each cycle produces sub-batches of biological material. In order to achieve process traceability in a continuous cell culture and harvest cycle, a sub-batch of the produced biological substance is first collected in a separate collector, the biological substance is then continuously directed from each collector through a downstream treatment device for downstream treatment of the biological substance to purify the biological substance at least into at least one batch containing the purified biological substance.

Description

[0001] The present invention relates to bioreactors and cell culture contents, and their use in large-scale preparation or production of biological substances (e.g., antibodies) by cell cultures cultivated and / or maintained in bioreactors. In the field of biological substance preparation, there is a difference between batch preparation and continuous preparation. In general, batch preparation involves producing biological substances by multiple separate process steps in a predetermined execution order, and generally involves some interruptions between continuous steps to test the quality of intermediate products. Therefore, in the entire production process, individual steps (especially bioreactors) may have a long residence time, and production efficiency and / or product output are relatively low due to product degradation over time and low efficiency of process steps. Typical batch processes last for 3 weeks.

[0002] In contrast, continuous production relies on the production of biomass using more automated systems that are able to produce and process biomass on a substantially continuous basis with little or no interruptions between process steps, and is therefore generally a more efficient production method.

[0003] In terms of quality control, batch and continuous manufacturing generally must meet the same or similar standards. Since continuous manufacturing is a more automated process, it is better suited to early detection of anomalies or problems and can prevent or minimize failures. However, compared to more conventional batch manufacturing, the current disadvantage of using continuous manufacturing processes to produce biological substances is traceability, that is, due to the continuous nature, the intermediate and final products of the continuous process cannot be easily and / or properly separated and linked to the cell culture batches allocated by the upstream process, including its raw materials, in process control and related deviations, especially the traceability methods of biological substances may not meet or comply with current regulations. Therefore, when an error occurs in the production line and causes a process failure, it may be necessary to stop the manufacturing process completely, resulting in unqualified products, product shortages and increased costs.

[0004] Therefore, it is an object to provide a method for the continuous preparation of biological substances that overcomes this disadvantage. To this end, it is also an object to provide a biopharmaceutical preparation system for the continuous preparation of biological substances. A specific object is to provide such a method and system, which can produce biological substances efficiently and cost-effectively, preferably by using cell culture with high cell density.

[0005] According to a first aspect, a method for continuously preparing a biomass is provided, the method using a bioreactor or a group of multiple bioreactors to cultivate mammalian cells in a culture medium to produce a batch of the biomass; the method includes performing multiple cell culture and harvest cycles, each cycle producing a sub-batch of the biomass, and the method includes the steps of: culturing cells, harvesting the culture medium containing the biomass while retaining the cells in the bioreactor, filtering the culture medium containing the biomass, and collecting the sub-batches of the biomass in a collector (e.g., a harvesting container); wherein, in the continuous cell culture and harvest cycles, the sub-batches of the biomass produced are collected in separate collectors, and wherein each sub-batch of the biomass and its associated raw materials under process control and deviation are continuously processed from each collector through a downstream processing device for downstream processing of the biomass to purify the biomass into at least one biomass batch containing purified biomass.

[0006] Although the definitions of batch, sub-batch and lot may vary slightly in the art, for example depending on differences between different regulatory agencies in different jurisdictions, the definitions are intended to be as follows. "Batch" means a specific quantity of a biological substance (e.g., a drug or other material) that is intended to have consistent characteristics and quality within specified limits and is produced according to a single preparation sequence within the same production cycle. "Sub-batch" means a portion of a specific quantity of a biological substance of a corresponding batch produced in one bioreactor during one culture and harvest cycle. "Batch" means a batch or a specific portion of a batch that has consistent characteristics and quality within specified limits; or, for a drug substance produced by a continuous process, a specific amount produced per unit time or quantity in a manner that ensures that it has consistent characteristics and quality within specified limits.

[0007] The problem of lack of traceability in continuous processes is that, taking into account the residence time distribution of the continuous process steps, it is not possible to provide the necessary separation of batches downstream of the process step that can be assigned to batches or sub-batches upstream of the process step. However, since the successive sub-batches of the biomass according to the first aspect of the present invention are collected in at least two separate collectors, the residence time distribution between these successive sub-batches in the downstream processing may be greatly reduced to no overlap at all, or at most some minimal overlap limited to the directly preceding and succeeding sub-batches. According to the first aspect of the present invention, the minimum overlap between the successive sub-batches is at most 5% (by volume) of the total volume of the sub-batch with the smallest total volume, but preferably the overlap (if any) does not exceed 3% (by volume) of the total volume of the sub-batch with the smallest total volume, and more preferably does not exceed 1% (by volume) of the total volume of the sub-batch with the smallest total volume.

[0008] This enables the separation of sub-batches from each collector in a limited and traceable downstream biomass batch. In the event of a process failure, relatively small amounts of defective material, such as one or a few batches or one or a few sub-batches, can be isolated, which generally results in less rejects and lower production costs for the biomass.

[0009] Preferably, the collection of the sub-batches of biological material is performed alternately with two collectors. Alternation between the two collectors provides the desired traceability with minimal equipment requirements for this purpose. More than two collectors can be used as required to further improve traceability, but at the expense of more equipment. Optionally, each sub-batch of biological material is collected in a purified form in a corresponding collector.

[0010] In a particular aspect of the method, in a continuous cell culture and harvest cycle, different or separate bioreactors are used to perform the steps of culturing cells, harvesting the culture medium containing biological substances while retaining cells in the bioreactor and filtering the culture medium containing biological substances, and each bioreactor is arranged to culture cells in the culture medium to produce corresponding batches of biological substances. Preferably, the continuous cell culture and harvest cycle is performed alternately with two bioreactors. The connection of multiple bioreactors with multiple collectors provides continuous preparation and supply of biological substance sub-batches, further improving production efficiency. For example, at least one bioreactor can keep running, to perform multiple cell culture and harvest cycles, and provide biological substance sub-batches to at least one collector for continuous downstream processing. One or more other bioreactors can be used as needed, for example, multiple cell culture and harvest cycles can be prepared to be performed using new or fresh cell cultures. Preferably, the bioreactor is controlled to provide a sub-batch to any collector within a desired time interval, for example, a first bioreactor can supply a sub-batch to a first collector within a first time interval, and a second bioreactor can supply a sub-batch to a second collector within a second time interval, wherein the first time interval and the second time interval are continuous, partially overlapped in time, or completely overlapped in time. Preferably, a corresponding collector is configured for each bioreactor to receive only a sub-batch of biomass from its corresponding bioreactor. However, two or more bioreactors can also be connected to a corresponding collector. For example, two or more relatively small bioreactors can be used to replace a relatively large bioreactor. For example, two or more bioreactors can each supply a certain amount of biomass or a sub-batch of biomass to a corresponding collector, wherein each amount or sub-batch is received continuously, partially overlapped, or simultaneously in the collector.

[0011] Optionally, in the method, the downstream processing of each biological material sub-batch includes a virus inactivation step and collecting each biological material sub-batch in a post-virus inactivation container. The collection of consecutive sub-batches of biological material in a post-virus inactivation container is preferably performed alternately with two separate post-virus inactivation containers. Optionally, the downstream processing of each biological material sub-batch includes at least one of a multi-column chromatography, a cation exchange, an anion exchange, a nanofiltration, a concentration and a diafiltration step.

[0012] In certain aspects, the method involves alternatingly performing any of the processing steps of multiple column chromatography, cation exchange, anion exchange, nanofiltration, concentration, and diafiltration on successive sub-batches in a dual setup apparatus configured to perform the corresponding one or more processing steps.

[0013] For a fully continuous production process, in a desired aspect, the method involves supplying successive sub-batches of biological material with two alternatingly controlled bioreactors to two connected collectors, from which the corresponding sub-batches are respectively and continuously processed through a dual set of downstream processes, wherein the downstream processing device is selected from a virus inactivation device and a post-virus inactivation container, a multi-column chromatography device, a cation exchange device, an anion exchange device, a nanofiltration device, a concentration device and a diafiltration device. The dual set of downstream processing devices can be provided in series or in parallel, and is preferably controllable for alternating the processing of successive sub-batches of biological material.

[0014] Preferably, in the method, the culturing and harvesting cycle comprises incubating the cell culture in the bioreactor until the cell culture reaches an early stationary phase or a stationary phase, and harvesting the medium containing the biological matter by separating the cells from the medium containing the biological matter, while retaining substantially all of the cells in the cell culture in the bioreactor for continued culturing with fresh medium.

[0015] Optionally, the cells used in the method are suspension cells, preferably CHO cells.

[0016] In certain aspects, the biological substance produced by the method is an antibody or an antigen-binding fragment thereof.

[0017] According to the purpose of the present invention, on the other hand, there is provided a biopharmaceutical preparation system for continuously preparing biological substances, comprising a bioreactor arranged to culture cells in a culture medium to produce batches of biological substances; wherein the bioreactor is connected to a separator, the separator is arranged to receive a continuous flow of a culture medium containing cells and produced biological substances from the bioreactor, and the separator is configured to retain the cells in the bioreactor and direct the produced biological substances to a collector (e.g., a harvesting container) of the system, the collector having an inlet connected to the separator for receiving a sub-batch of biological substances from the bioreactor, and having an outlet connected to a downstream processing device of the system for continuously downstream processing the sub-batches into at least one batch of biological substances; wherein the system comprises at least one additional collector, and wherein the collector and the at least one additional collector are configured to alternately receive sub-batches of biological substances and flow the corresponding sub-batches through the downstream processing device.

[0018] The biopharmaceutical preparation system lends itself to being particularly well suited for carrying out the methods according to one or more aspects described herein. Specifically, providing at least two collectors enables the system to collect successive subbatches of biological material in at least two separate collectors and to perform successive downstream processing with a substantially reduced overlap in residence time distributions between successive subbatches. Thus, the system is capable of separating subbatches from each collector in a restricted and traceable downstream batch of biological material.

[0019] Preferably, the system comprises at least two bioreactors, each of which is arranged to culture cells in a culture medium to produce a corresponding batch of biomass. Preferably, continuous cell culture and harvest cycles are performed alternately with two bioreactors. Continuous cell culture and harvest cycles can be performed at least partially simultaneously, but preferably the steps of collecting sub-batches of biomass into collectors in each cycle are separated at least in time. The connection of multiple bioreactors with multiple collectors provides continuous preparation of biomass, further improving production efficiency. For example, at least one bioreactor can be kept running to perform multiple cell culture and harvest cycles, and provide sub-batches of biomass to at least one collector for continuous downstream processing. One or more other bioreactors can be used as needed, for example, multiple cell culture and harvest cycles can be prepared to be performed with new or fresh cell cultures. Preferably, the bioreactor is controlled to provide sub-batches to one of the collectors within a desired time interval, for example, a first bioreactor can supply sub-batches to a first collector within a first time interval, and a second bioreactor can supply sub-batches to a second collector within a second time interval, wherein the first time interval and the second time interval are continuous, partially overlapped in time, or completely overlapped in time.

[0020] Optionally, at least two bioreactors are connected to the collector and at least one further collector such that respective sub-batches of biomass from each bioreactor flow to the at least one collector.

[0021] In a particular aspect, the system comprises control means arranged to control the flow of sub-batches of biomass from each bioreactor to any one of the collectors.

[0022] Preferably, the downstream processing means of the system comprises at least two post-virus inactivation containers arranged to alternately collect successive sub-batches of biological matter from the at least one collector.

[0023] Specifically, the downstream processing device in the system includes at least one of the following: a multi-column chromatography device, a cation exchange device, an anion exchange device, a nanofiltration device, a concentration device and a diafiltration device, wherein at least one device is provided in a dual setting and is configured to alternately perform corresponding one or more processing steps on consecutive sub-batches of biological material received by any collector.

[0024] For a fully continuous production process, in a desired aspect, the system comprises two alternatingly controlled bioreactors for supplying continuous sub-batches of biological material, the two alternatingly controlled bioreactors being connected to two separate collectors, each collector being connected to a respective separate downstream processing system for causing the respective sub-batches to flow continuously through the respective downstream processing system, each downstream processing system comprising at least one dual arrangement of downstream processing devices; wherein the downstream processing devices are selected from a virus inactivation device and a post-virus inactivation container, a multi-column chromatography device, a cation exchange device, an anion exchange device, a nanofiltration device, a concentration device, and a diafiltration device. The dual arrangement of downstream processing devices can be provided in series or in parallel, and is preferably controllable for alternatingly processing continuous sub-batches of biological material.

[0025] In one aspect, the method and system described herein can relate to large-scale cultivation or fermentation of cell culture, preferably with high-density cells. The advantage of the method and system of the present invention is that the output of biological substances is high. Along with each further incubation / cultivation and harvesting cycle, the output of material further increases. Therefore, the method of the present invention preferably includes at least 2 times, more preferably at least 5 times, more preferably at least 10 times and preferably at least 20 incubation and harvesting cycles.

[0026] The cell culture is preferably harvested with a harvesting system that maintains many conditions of the incubation step. Therefore, the harvesting system preferably includes a device for maintaining the culture temperature. When the volume of the cell culture in the bioreactor exceeds the capacity of the harvesting system, the entire cell culture can still be harvested. Preferably, this is done by feeding purified cell culture aliquots to the harvesting system in sequence. When substantially the entire cell culture has been processed, the harvesting cycle is completed. The cell culture waiting to be processed will not be mixed with cells that have already been processed by the harvesting system. This is achieved by feeding the processed cells to different bioreactors. Therefore, fresh culture medium is provided to the processed cells and fed to different bioreactors for subsequent incubation and harvesting cycles. If necessary, the cell density can be adjusted at this stage. Cells can be concentrated or diluted according to the operator's wishes. An advantage of the present invention is that a harvesting system with a capacity different from the cell culture volume can be implemented.

[0027] It is desirable to increase cell density, particularly in the first cycle, to quickly achieve the target cell density required for production of the biomass. Dilutions are often required in subsequent cycles to accommodate continued growth in cell number.

[0028] The biological substance is preferably a substance secreted by the cell. The biological substance is preferably a protein, preferably a secretory protein. The protein preferably contains more than 50, and preferably more than 100 amino acids. In a particularly preferred embodiment, the biological substance is a soluble form of a growth factor, an antibody or a membrane-bound ligand. The biological substance may also include an antigen-binding fragment of an antibody. Non-limiting examples of such fragments are single-chain Fv fragments, FAB fragments, monovalent antibodies and heavy chain antibodies only. In a preferred embodiment, the antibody is a mammalian antibody. In a preferred embodiment, the antibody is a monoclonal antibody. In a preferred embodiment, the antibody is a mouse antibody, a human antibody or a mouse / human chimeric antibody. The chimeric antibody is preferably a humanized mouse monoclonal antibody or a human antibody. There are different types of humanization. Typically, at least the constant portion of the antibody is human.

[0029] Modern humanized antibodies also contain human variable regions, in which the CDR regions are replaced by mouse CDRs. Alternatively, the antibody contains mutations in the mouse variable regions to remove predicted human B cell and / or T cell epitopes. Currently, so-called fully human monoclonal antibodies can be generated. These are completely generated from human sequences. However, immune responses to these fully human antibodies are still possible, depending on the mode of administration and the nature of the CDRs.

[0030] Cells can be grown in suspension culture or adherent culture. Some cells grow naturally in suspension, not attached to the surface, such as cells present in blood. Some cell lines are also modified to survive in suspension culture, so they can grow to a higher density than that allowed by adherent conditions. Adherent cells require a surface, such as tissue culture plastics or microcarriers, which may be coated with extracellular matrix components to increase adhesion properties and provide other signals required for growth and differentiation. Attached cells can generally be suitable for suspension growth, alternatively the cells can be adhered to microcarriers as described above, then suspended and cultured in a bioreactor. The cells in the present invention are preferably cells grown in suspension. Preferred mammalian cells. Preferred production cells are Chinese hamster ovary or CHO cells and NSO cell lines. In a particularly preferred embodiment, the cell is a CHO cell.

[0031] Cell growth in a typical fed-batch culture of mammalian cells can be divided into seven phases: (A) lag phase; (B) early log phase; (C) log / exponential phase; (D) early stationary phase; (E) stationary phase; (G) early death phase; and (F) death phase.

[0032] A. During the lag phase, cells adapt themselves to growth conditions. The number of cells in the population does not increase.

[0033] B. During the early log phase, an increasing number of cells have adapted and entered the exponential phase.

[0034] C. The exponential phase (sometimes called the logarithmic phase or logarithmic growth phase) is a period characterized by cell doubling. The number of new cells that appear per unit time is proportional to the number of existing cells. If growth is unlimited, doubling will continue at a constant rate, so the number of cells and the population growth rate will double in each successive period of time. For this type of exponential growth, plotting the natural logarithm of the number of cells against time will produce a straight line. The slope of this line is the specific growth rate of the cells. The actual rate of this growth (i.e., the slope of the line in the graph) depends on the growth conditions and the cell type.

[0035] D. The early stable phase (or early plateau phase) is the period between the logarithmic phase and the stable phase, characterized by a gradual decline in the population growth rate. This period is characterized by a population growth rate that is one-third or less of the maximum growth rate in the exponential phase. The early stable phase ends when the growth of the cell population is zero or less.

[0036] E. The stationary phase or plateau phase is usually due to growth limiting factors; primarily nutrient depletion and / or inhibition of product formation. During the stationary phase, the size of the cell population remains more or less constant. The production of biomass is usually highest during this period.

[0037] F. During the decline phase, cells run out of nutrients, or toxic products accumulate to a certain level, leading to cell death.

[0038] G. Early apoptosis defines the end of the stationary phase, when increasing numbers of cells die.

[0039] According to the treatment mode of the cell before starting to cultivate, there may be or may not be a lag phase. For example, large-scale cultivation usually starts from seed culture and pre-culture. In this case, there may be a very short lag phase or there may not be a lag phase, because the cell may have adapted to the culture conditions of the fed-batch culture in the appropriate seed culture and / or pre-culture. In a circulation culture system, such as the present invention, the following circulation repeats the C and D periods usually. Alternatively, one or more circulations include steps E, F and G in addition. The exponential phase of the subsequent circulation may or may not reach the maximum growth rate identical with the first circulation. In each circulation, the beginning of the early stable phase is characterized by: the population growth rate drops to one-third or lower from the maximum growth rate of the previous exponential phase. The growth rate is usually calculated according to the tangent of the growth curve.

[0040] In a particularly preferred embodiment, the culture of each cycle is harvested when the culture is in phase C, D, or E. Preferably, the culture of each cycle is harvested when the culture is in phase C or D, more preferably in phase D. In another embodiment, the cultures of cycle 2 and beyond are harvested after at least one day, preferably at least two days, more preferably at least three days of incubation, regardless of which phase of growth the cells are in when the harvesting step is initiated.

[0041] In another embodiment, the culture of each cycle is harvested when a predetermined cell density is reached. For the culture after the first culture and harvest cycle, the culture is preferably harvested when the cells in the culture reach a cell density of at least 0.5×10e8, preferably at least 1×10e8, more preferably at least 2×10e8 per ml.

[0042] Figure 1 and Figure 2 An example of a system for producing a biomass is given. In this system, cells that produce the biomass are cultured and separated from a culture medium containing the biomass. After separation, preferably substantially all of the cell feed is returned to the bioreactor to continue the culture with fresh culture medium. Figure 1In the system 1, a plurality of bioreactors 10, 15 are provided. The bioreactors are arranged to incubate cell cultures of cells suspended in a culture medium in the bioreactors. Each bioreactor 10, 15 includes a culture medium feed inlet 16, which is arranged to feed culture medium or its components into the corresponding bioreactor to compensate for outflow losses. Specifically, the feed inlet 16 can be directly connected to the bioreactor, or can be connected to a pump and a switching unit, which is connected to a separator 30 of the system. In addition, a device for detecting cell count or density in the bioreactors 10, 15 can also be provided. Typically, such a device can be a cell counting detector, a densitometer or a flow cytometer of a type known in the art. The detector can be arranged to detect cell density. A predetermined threshold can be determined heuristically or by calculation to be input to the detector, indicating that the incubation of the cell culture in one of the bioreactors has reached an early stable phase or a stable phase as described above. The separators 30, 35 are arranged in communication between the bioreactor and the harvesting filtration module 20. Thus, each bioreactor 10, 15 is connected to a harvest filtration module 20 via a respective separator 30, 35. Each separator 30, 35 comprises a hollow fiber perfusion filter supplied with a cell culture, which is pumped out of the respective bioreactor 10, 15 with a pump to separate the permeate containing the product from the cells of the cell culture. The cells remain in the respective bioreactor, while the permeate containing the product is continuously filtered by the membrane filtration device 21, 22 and collected in the respective collector 23, 24 of the harvest filtration module 20. Each cultivation and harvesting cycle performed in one of the bioreactors 10, 15 in this way provides a biomass sub-batch in the respective collector 23, 24. The collector preferably comprises a harvest bag 23, 24 or consists of a harvest bag 23, 24. Each collector 23, 24 is fluidly connected to a downstream processing system 40 for continuous downstream processing of the biomass sub-batch into a batch 50, 55 of biomass. Downstream treatment system 40 comprises one or more treatment devices (not shown) selected from the following: virus inactivation device and virus inactivation back container, multi-column chromatography device, cation exchange device, anion exchange device, nanofiltration device, concentrating device and diafiltration device. One or more downstream treatment devices can be provided with dual settings, such as in series or in parallel. As shown in the figure, the preferred setting of this system comprises a dual setting bioreactor (10, 15) for providing a biomass sub-batch, a dual setting collector (23, 24) for receiving a biomass sub-batch from a corresponding bioreactor, a dual setting holding container (41, 42) for accommodating a partially processed biomass sub-batch from a corresponding collector, and a dual setting storage container (50, 55) for storing a batch of biomass. The system may include an additional holding container for a dual setting for each or any treatment step.

[0043] In a preferred embodiment of the present invention, during the incubation of cell culture in a bioreactor, at least a portion of the cell culture flows continuously into a separation system with a size selective membrane. However, other separation devices may be used alternatively or additionally. After separation, the cells are preferably retained in the bioreactor to carry out additional culture and harvest cycles, but the cells may also be optionally transferred to another bioreactor. With the latter arrangement, a harvest system with a fluid capacity less than the volume of the cell culture may be realized. In further incubation, preferably at least a portion of the cell culture flows continuously into a separation system with a size selective membrane. In the separation device, the consumed culture medium and the material (waste) with a molecular weight less than that of the biological substance are removed from the culture. In the harvest system, the consumed culture medium, biological substance and the material (waste) with a molecular weight greater than that of the biological substance, such as cell debris, nucleic acid and protein, are taken out. This preferred embodiment is a particularly effective way, which provides conditions for multiple culture and harvest cycles at very high cell density, while maintaining a relatively low-cost system associated with continuous cultivation in a bioreactor, wherein at least a portion of the cell culture flows continuously into a separation system with a size selective membrane.

[0044] Preferably, the culture conditions during the culture are maintained in the separation system. The cultured cells undergo culture conditions in each culture and harvest cycle of the present invention. From the perspective of the cells, the culture is a continuous culture, while from the perspective of the process operator responsible for handling waste disposal and biomass harvesting, the culture is semi-continuous. From the perspective of the cells, a system including a separation system, a centrifuge and one or more bioreactors can be regarded as a bioreactor for culturing cells. The cells undergo culture conditions throughout the program. Not all culture conditions must be maintained. The culture condition preferably maintained is temperature, and another culture condition preferably maintained is oxygenation. Another culture condition preferably maintained is pH. Maintaining culture conditions preferably means that the deviation of the conditions throughout the program from the condition values ​​at the beginning of the program does not exceed 20%. The temperature is preferably maintained at 35.5°C to 37.5°C, preferably at 37°C. The temperature can be reduced to a second temperature from a first temperature during the culture of cells to a target cell density to induce a stable biomass production rate by continuous culture medium perfusion feeding. Oxygenation is preferably maintained at 40% to 60% (expressed as dissolved oxygen concentration), preferably at 50% dissolved oxygen concentration, and pH is preferably maintained at a value between pH 6.7 and 7.5, preferably at pH 7 or 7.1.

[0045] An advantage of the methods and systems according to one or more aspects and embodiments described herein is that the ability to separate continuously harvested material in two or more separate collectors allows for continuous downstream processing while maintaining some batch-like traceability. The biomass can be continuously flowed from the respective collectors to feed further downstream processing systems. By processing in this manner, high product yields can be achieved in a low-volume continuous operation mode.

Claims

1. A method for continuously preparing a biomass, the method using a bioreactor to cultivate mammalian cells in a culture medium to produce a batch of the biomass; the method comprising performing a plurality of cell cultivation and harvesting cycles, each cycle producing a sub-batch of the biomass, and comprising the steps of: cultivating cells, harvesting the culture medium containing the biomass while retaining the cells in the bioreactor, filtering the culture medium containing the biomass, and collecting the sub-batches of the biomass in a collector, such as a harvesting vessel; wherein in the continuous cell cultivation and harvesting cycles, the sub-batches of the biomass produced are collected in separate collectors, and wherein each sub-batch of the biomass is continuously directed from each collector through a downstream processing device for downstream processing of the biomass to purify the biomass into at least one biomass batch containing purified biomass.

2. The method according to claim 1, wherein the collecting of the subbatches of biomass is performed alternately with two collectors.

3. The method according to claim 1 or 2, wherein each subbatch of biomass is collected in purified form in a respective collector.

4. A method according to any one of the preceding claims, wherein the steps of culturing cells, harvesting the culture medium containing the biomass while retaining the cells in the bioreactor, and filtering the culture medium containing the biomass are performed in a continuous cell culturing and harvesting cycle using separate bioreactors, each bioreactor being arranged to culture cells in a culture medium to produce a respective batch of biomass.

5. The method according to claim 4, wherein continuous cell culture and harvesting cycles are performed alternately using two bioreactors.

6. The method according to any one of the preceding claims, wherein the downstream processing of each biomass subbatch comprises a viral inactivation step and collecting each biomass subbatch in a post-viral inactivation container.

7. The method according to claim 6, wherein collecting successive subbatches of biological material in the post-virus inactivation container is performed alternately using two separate post-virus inactivation containers.

8. The method of any one of the preceding claims, wherein downstream processing of each biomass sub-batch comprises at least one of multi-column chromatography, cation exchange, anion exchange, nanofiltration, concentration and diafiltration.

9. The method of claim 8, wherein any of the processing steps of multiple column chromatography, cation exchange, anion exchange, nanofiltration, concentration and diafiltration on consecutive sub-batches is performed alternately in a dual setup apparatus configured to perform the corresponding one or more processing steps.

10. The method of any one of the preceding claims, wherein a culture and harvest cycle comprises incubating a cell culture in a bioreactor until the cell culture reaches early stationary phase or stationary phase, and harvesting the culture medium containing the biological matter by separating the cells from the culture medium containing the biological matter, while retaining substantially all of the cells in the cell culture in the bioreactor for continued culture with fresh culture medium.

11. The method according to any one of the preceding claims, wherein the biological agent is an antibody or an antigen-binding fragment thereof.

12. A biopharmaceutical preparation system for continuously preparing a biological substance, comprising a bioreactor arranged to culture cells in a culture medium to produce a batch of biological substance; wherein the bioreactor is connected to a separator, the separator being arranged to receive a continuous flow of a culture medium containing cells and produced biological substance from the bioreactor, and the separator being configured to retain the cells in the bioreactor and to flow the produced biological substance to a collector of the system, such as a harvesting container, the collector having an inlet connected to the separator for receiving a sub-batch of biological substance from the bioreactor, and having an outlet connected to a downstream processing device of the system for continuously downstream processing the sub-batch into at least one batch of biological substance; wherein the system comprises at least one additional collector, and wherein the collector and the at least one additional collector are configured to alternately receive the sub-batches of biological substance and process the corresponding sub-batches by the downstream processing device.

13. The system of claim 12, comprising at least two bioreactors, each bioreactor being arranged to culture cells in a culture medium to produce a respective batch of biomass.

14. The system of claim 13, wherein the at least two bioreactors are connected to the collector and at least one further collector such that a respective sub-batch of biomass from each bioreactor flows into the at least one collector.

15. A system according to claim 14, comprising control means arranged to control the flow of sub-batches of biomass from each bioreactor to any one of the collectors.

16. The system according to any one of the preceding claims, wherein the downstream processing device comprises at least two post-virus inactivation containers arranged to alternately collect consecutive sub-batches of biological material from at least one collector.

17. A system according to any of the preceding claims, wherein the downstream processing device includes at least one of a multi-column chromatography device, a cation exchange device, an anion exchange device, a nanofiltration device, a concentration device and a diafiltration device, wherein at least one device is provided in a dual setting and configured to alternately perform corresponding one or more processing steps on consecutive sub-batches of biological matter received by any one collector.

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