Process and system for producing inoculum

Abstract

A process and system for producing inoculum for downstream cell production is disclosed. The inoculum is produced in a perfusion bioreactor in which nutrient media feed is increased as biomass concentration within the bioreactor increases. A biomass sensor can be used to monitor biomass concentration periodically or continuously. This information can be supplied to a controller for automatically increasing the nutrient media feed rate in direct proportion to the production of inoculum with increasing cell density. The process and system can also include an automated subsystem for maintaining a constant volume level within the perfusion bioreactor during the process.

Description

Technical Field

[0001] Typically, this disclosure relates to an inoculation process that produces a rapid increase in cell density and viable cell count during culture. Background Technology

[0002] Bioreactors are widely used in the biopharmaceutical industry. A bioreactor is a device that can conduct biological reactions or processes on a laboratory or industrial scale. Bioreactors can be used to produce all different types of biological products. Biological products can include, for example, cell cultures and materials derived from cell cultures, including beverages, biofuels, bioenergy, biochemicals, antibiotics, amino acids, enzymes, monoclonal antibodies, monomers, proteins, food cultures, biopolymers, alcohols, flavorings, fragrances, etc. In some embodiments, cell cultures can be grown for cell therapy. Cell therapy is the prevention, treatment, cure, or relief of disease or injury in a person by administering autologous, allogeneic, or xenogeneic cells that have been manipulated or altered in vitro. One goal of cell therapy is to repair, replace, or restore damaged tissues or organs.

[0003] Cell cultures are typically grown in batch processes, where the biological material remains in the bioreactor until the reaction time is complete. In some of these processes, the fluid culture medium contained within the bioreactor can be periodically or continuously removed and re-fed to replenish nutrients contained in the fluid medium and to potentially remove harmful byproducts generated during the process.

[0004] Before cell cultures can be grown in a batch reactor as described above, an inoculation process must first be performed. For example, an inoculum of microorganisms is needed to provide a population with the desired viable cell count, suitable for scale-up to a level suitable for commercial-scale production. Currently, the inoculation process is carried out in batch mode. Due to nutrient limitations and the accumulation of inhibitory metabolites, there are limitations on the maximum cell density compared to these conventional processes. Lower cell densities during the inoculation process can lead to longer time cycles required in commercial batch reactors. For example, feeding an inoculum to a batch reactor with a low cell density requires downstream production bioreactors to first spend a significant amount of time generating cell clusters, rather than producing the desired bioproducts, such as proteins. Ultimately, longer incubation times are required in larger commercial batch reactors, directly impacting the efficiency of the overall process.

[0005] In light of the above, there is a need for improved methods and systems for producing microbial inoculum for subsequent transfer to larger production bioreactors. There is also a need for methods and systems for obtaining inoculum that can achieve higher viable cell counts and produce higher cell densities during the inoculation process. Furthermore, there is a need for an inoculation process that can feed the inoculum into the production bioreactor and reduce the amount of time required to produce bioproducts within the production bioreactor. Summary of the Invention

[0006] Typically, this disclosure relates to an inoculation process that produces a rapid increase in cell density and viable cell count during culture. The inoculation process and system of this disclosure typically include a perfusion bioreactor, wherein the perfusion rate varies and increases with the increase of cell culture biomass, rather than remaining constant. In fact, the nutrient feed rate to the perfusion bioreactor can be regulated using automated methods based on real-time biomass measurements. Through this process, inoculum with a significantly increased cell density and / or viable cell count can be produced. The inoculum can then be fed into a larger production bioreactor for the production of bioproducts, such as proteins. With the inoculation process and system of this disclosure, the amount of time required in the production bioreactor can be significantly reduced, which can significantly increase the productivity of the process. We have found that the control of the perfusion rate is improved when based on measurements of viable cell volume / biovolume, in contrast to previous methods based on viable cell density.

[0007] Therefore, in a first aspect, this disclosure relates to a process for producing an inoculum for subsequent cell culture production processes, comprising:

[0008] Introduce cell cultures into a perfusion bioreactor;

[0009] Nutrient media are fed into the perfusion bioreactor at a certain flow rate and fluid media are removed from the perfusion bioreactor;

[0010] A biomass sensor, connected to a controller, is used to determine the biomass concentration over time within the perfusion bioreactor; and

[0011] The controller is configured to adjust the flow rate of the nutrient medium entering the perfusion bioreactor based on the biomass concentration sensed by a biomass sensor.

[0012] The flow rate of the nutrient medium is adjusted based on the following relationship:

[0013]

[0014] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0015] This is a biovolume fraction and refers to the volume of a perfusion bioreactor within the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0016] P is the infusion rate expressed as mL feed / mL bioreactor / day.

[0017] In related embodiments, the present invention also relates to a process for producing inoculum for subsequent cell culture production processes, comprising:

[0018] Introduce cell cultures into a perfusion bioreactor;

[0019] Nutrient media are fed into the perfusion bioreactor at a certain flow rate and fluid media are removed from the perfusion bioreactor;

[0020] A biomass sensor, connected to a controller, is used to determine the biovolume fraction over time within the perfusion bioreactor; and

[0021] The flow rate of the nutrient medium entering the perfusion bioreactor is adjusted based on the biovolume fraction. The controller is configured to adjust the flow rate based on information received from biomass sensors.

[0022] The flow rate of the nutrient medium is adjusted based on the following relationship: As described above.

[0023] This process can be used to produce inoculum for subsequent cell culture production methods, such as the production of bioproducts of interest. The process may include introducing cell cultures into a perfusion bioreactor. Nutrient media can be fed into the perfusion bioreactor at a given flow rate. Simultaneously, fluid media can be extracted from the perfusion bioreactor. The fluid media extracted from the perfusion bioreactor may also be filtered to prevent cell loss. During cell culture growth in the perfusion bioreactor, a biomass sensor connected to the cell culture fluid in the perfusion bioreactor determines the biomass concentration over time. The biomass sensor may also be connected to a controller. The nutrient media flow rate can then be adjusted based on the biomass concentration transmitted by the biomass sensor. The controller can be configured to adjust the media flow rate based on information received from the biomass sensor.

[0024] For example, the biomass sensor may include a capacitive sensor suitable for placement within a perfusion bioreactor. Alternatively, the biomass sensor may include an optical cell counter. In one embodiment, for example, the perfusion bioreactor may be in fluid communication with an automated sampling system. The automated sampling system may continuously or periodically remove samples from the perfusion bioreactor for testing using the biomass sensor. In one embodiment, the biomass sensor may acquire biomass concentration readings at least every 6 hours, such as at least every 4 hours, such as at least every 30 minutes, such as at least every 10 minutes. The biomass concentration measurements may be supplied to a controller, which may include an algorithm for determining the nutrient medium flow rate. For example, the nutrient medium flow rate may be adjusted based on the following relationship:

[0025]

[0026] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0027] This is a biovolume fraction and refers to the volume of a perfusion bioreactor within the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0028] P is the infusion rate expressed as mL feed / mL bioreactor / day.

[0029] Typically, the controller can be configured to increase the flow rate of the nutrient medium entering the perfusion bioreactor when the cell density within the bioreactor increases.

[0030] In addition to controlling the flow rate of the nutrient medium fed into the perfusion bioreactor, the flow rate of the fluid medium extracted from the perfusion bioreactor can also be controlled. For example, in one embodiment, the amounts of biomass and fluid medium within the perfusion bioreactor can be determined. Based on this amount, the rate at which the fluid medium is extracted from the perfusion bioreactor can be selectively increased or decreased. In one embodiment, for example, the amounts of biomass and fluid medium within the perfusion bioreactor can be determined using a weighing device. This weighing device can be communicated with a controller. Based on the weight information from the weighing device, the controller can be configured to control a pumping device in fluid communication with the perfusion bioreactor to selectively increase or decrease the extraction rate of the fluid medium. For example, the extraction rate of the fluid medium can be such that the volume of fluid medium and cell culture in the perfusion bioreactor remains constant during the process.

[0031] Instead of weighing devices, the process and system can also use volumetric level indicators to determine the amount of fluid medium within the perfusion bioreactor. The volumetric indicator can also be connected to a controller to automatically control the rate at which the fluid medium is removed from the perfusion bioreactor.

[0032] Perfusion bioreactors typically have volumes of approximately 10 liters to approximately 4000 liters, or approximately 1000 liters to approximately 4000 liters. During the inoculation process, cell cultures can reach a density greater than approximately 10 × 10⁻⁶ within the perfusion bioreactor. 6 Cells / mL, such as greater than about 30 × 10⁻⁶ 6 Cells / mL, such as greater than about 50 × 10⁻⁶ 6 Cells / mL, such as greater than about 70 × 10⁻⁶ 6 Cell density per mL. In one embodiment, the cell culture can reach 100 × 10⁶ cells / mL. 6 Cell density of cells / mL or higher. Cell density is typically less than approximately 1000 × 10⁶ cells / mL. 6 Cells / mL. Cell cultures can be maintained in a perfusion bioreactor for approximately 3 to approximately 12 days.

[0033] The process disclosed herein may further include the step of transferring the cell culture from the perfusion bioreactor to a second bioreactor after the desired cell density has been reached. For example, the second bioreactor may be a batch-feed reactor and may have a volume of about 10 L to about 30,000 L. For example, the volume of the second bioreactor may be larger than that of the perfusion bioreactor. For example, the ratio between the volume of the perfusion bioreactor and the volume of the second bioreactor may be about 1:3 to about 1:40, such as about 1:4 to about 1:10. The cell culture may be held in the second bioreactor for a time period of less than about 12 days, such as less than about 11 days, such as less than about 10 days, and may still be able to produce the desired amount of bioproduct.

[0034] This disclosure also relates to a system for producing inoculum for subsequent cell culture production processes. Therefore, in a second aspect, the invention further relates to a system for producing inoculum for subsequent cell culture production processes, comprising:

[0035] Infusion bioreactor;

[0036] Nutrient medium feed is in fluid communication with the perfusion bioreactor and is used to feed nutrient medium into the perfusion bioreactor to enable cell culture growth.

[0037] Used to remove the effluent of the fluid medium from the perfusion bioreactor;

[0038] A pumping device connected to the effluent from the perfusion bioreactor to remove a controlled amount of fluid medium from the perfusion bioreactor;

[0039] Weighing device used to monitor the weight of a perfusion bioreactor;

[0040] Biomass sensors, such as capacitive sensors, are in fluid communication with the perfusion bioreactor to determine the biomass concentration within the perfusion bioreactor; and

[0041] A controller connected to the biomass sensor and weighing device is configured to control the nutrient medium feed based on information received from the biomass sensor to increase or decrease the flow rate of the nutrient medium fed into the perfusion bioreactor. The controller is also configured to control the pumping device based on information received from the weighing device to increase or decrease the flow rate of the fluid medium extracted from the perfusion bioreactor.

[0042] The controller controls the flow rate of the nutrient medium entering the perfusion bioreactor based on the following relationship: As described above.

[0043] The system includes a nutrient medium feed in fluid communication with a perfusion bioreactor. The nutrient medium feed is used to introduce nutrient media into the perfusion bioreactor to promote cell culture growth. The perfusion bioreactor may also include an effluent for removing the fluid medium from the perfusion bioreactor. A pumping device may be communicated with the effluent to control the outflow of the fluid medium from the perfusion bioreactor. The system may further include a weighing device for monitoring the weight of the perfusion bioreactor and a biomass sensor communicated with the perfusion bioreactor for determining the biomass concentration within the perfusion bioreactor. A controller may be communicated with the biomass sensor and the weighing device. The controller may be configured to control the nutrient medium feed to increase or decrease the flow rate of the nutrient medium fed into the perfusion bioreactor based on information received from the biomass sensor. The controller may also be configured to control the pumping device to increase or decrease the flow rate of the fluid medium from the perfusion bioreactor based on information received from the weighing device. For example, the controller may include one or more microprocessors.

[0044] As described above, the processes and systems disclosed herein are particularly suitable for producing inoculum for subsequent cell culture production processes.

[0045] Therefore, in a third aspect, the present invention relates to a cell culture production process comprising:

[0046] Inoculum comprising host cells expressing biological products is produced by the method of the first aspect of the present invention;

[0047] Inoculation materials are introduced into production bioreactors, and cell cultures are introduced into perfusion bioreactors;

[0048] Culture host cells to produce biological products;

[0049] Harvesting biological products from cell cultures; and

[0050] Optionally, one or more purification steps may be performed on the biological product.

[0051] However, alternatively, the processes and systems of this disclosure can be used to produce bioproducts without transferring them to subsequent batch bioreactors. For example, in one embodiment, cell cultures can be incubated in a perfusion bioreactor to achieve a desired cell density. The cell cultures can then be fed into a purification process and / or into a process for harvesting bioproducts from the cell cultures.

[0052] Other features and aspects of this disclosure are discussed in more detail below. Attached Figure Description

[0053] The full and implementable disclosure of this invention is set forth in more detail in the remainder of the specification (including with reference to the accompanying drawings), wherein:

[0054] Figure 1 This is an embodiment of a perfusion bioreactor system for producing inoculum for downstream cell production according to the present disclosure;

[0055] Figure 2 This is an embodiment of a system for producing inoculum and transferring the inoculum to a large-scale bioreactor for producing bioproducts; and

[0056] Figure 3 It is a graphic representation illustrating the various benefits and advantages disclosed herein.

[0057] Figures 4A to 4B An illustrative example of variable feed according to embodiments of this document is shown.

[0058] Figure 5 The concentrated nutrient feed rate is shown as a function of the perfusion medium feed rate normalized per biological volume.

[0059] Figure 6 The concentrated nutrient feed rate is shown as a function of the per-cell standardized perfusion medium feed rate.

[0060] Figure 7 The concentrated nutrient feed rate is shown as a function of the perfusion medium feed rate normalized per biological volume.

[0061] Figure 8 The concentrated nutrient feed rate is shown as a function of the per-cell standardized perfusion medium feed rate.

[0062] Figure 9 A constant per-biovolume feeding strategy based on embodiments herein is illustrated.

[0063] Figure 10The selection of conditions for examining the effect of biovolume ratio perfusion rate on cell culture performance is shown.

[0064] Figures 11A to 11B Media consumption and cell density under four BVSPR conditions are shown.

[0065] Figure 12 The cell density of five cell clones cultured under BVSPR conditions of 7 mL / mL / day is shown.

[0066] Figure 13 The N-1 process is shown scaled up from laboratory scale to a 50L bioreactor. Detailed Implementation

[0067] Those skilled in the art will understand that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of this disclosure.

[0068] Generally, this disclosure relates to processes and systems for producing bioproducts. More specifically, this disclosure relates to processes and systems for producing inoculum, which will be transferred to a large-scale bioreactor for producing bioproducts. The inoculum is grown in a perfusion bioreactor, wherein the purpose of the process is to produce a rapidly increasing amount of biomass during cultivation. Process control is used to carefully control the rate of nutrient medium feed into the perfusion bioreactor and the rate of fluid medium removal from the perfusion bioreactor. According to this disclosure, the nutrient medium flow rate and removal flow rate are periodically and / or constant to maintain an optimal feed rate per unit of biomass within the perfusion bioreactor while maintaining constant volume or constant mass conditions. In one embodiment, the process can be fully automated to adjust the nutrient medium feed rate based on real-time biomass concentration measurements. For example, the nutrient medium feed rate can be adjusted such that it is proportional to the current amount of biomass in the reactor.

[0069] In addition to producing inoculum, the processes and systems of this disclosure can also be used to produce cell cultures that do not require further incubation time. For example, the perfusion bioreactor of this disclosure can produce cell cultures with cell density, wherein the bioproduct can be harvested directly from the perfusion bioreactor. Alternatively, the cell cultures incubated in the perfusion bioreactor can be fed into a purification process for subsequent harvesting of the bioproduct.

[0070] The methods and systems disclosed herein can be applied to any suitable cell culture product. For example, the methods of this disclosure are particularly suitable for the production of biopharmaceuticals such as biotherapeutic proteins. For example, biotherapeutic proteins are generated from genetically modified mammalian cells. In one embodiment, the cell culture can be produced via recombinant gene expression in a cell host. Such production can originate from established cell lines of culture, such as, for example, CHO, NSO, or PER.C6. These cells can express the protein of interest and subsequently secrete the protein into a medium. However, it should be understood that the processes and techniques of this disclosure are not limited to protein production, and any suitable cell culture can be subjected to the controls described herein.

[0071] As described above, in one embodiment, this disclosure generally relates to systems and processes for producing inoculum that can be transferred to a larger bioreactor, such as a commercial-scale bioreactor. According to this disclosure, inoculum containing microorganisms in a compatible state and well-suited for further growth in a production bioreactor at relatively high cell densities and viable cell counts can be prepared. For example, the processes and systems of this disclosure can achieve high levels of viable material under suitable physiological conditions for use as inoculum. In addition to protein production, the processes and systems of this disclosure can be used to produce antimicrobial agents, enzymes, beverages, pharmaceuticals, toxins, vitamins, amino acids, etc.

[0072] refer to Figure 1 An embodiment of a perfusion bioreactor system that can be used to generate inoculum according to the present disclosure is illustrated. Figure 1 The diagrams shown are for illustrative purposes only and do not limit the types of perfusion bioreactor systems that can be used to generate quality attribute information. Typically, perfusion bioreactor systems can be configured as highly automated process development platforms. Using perfusion bioreactor systems, inoculum with very high cell densities and / or viable cell counts can be produced.

[0073] like Figure 1 As shown, the perfusion bioreactor system includes a perfusion bioreactor 10. The perfusion bioreactor 10 can contain any suitable bioreactor, depending on the cell culture being propagated. For example, the perfusion bioreactor 10 can include a fermenter, a stirred tank reactor, a wave reactor, a shaking reactor, etc. Figure 1 The perfusion bioreactor 10 in the illustrated embodiment comprises a hollow vessel or container including a bioreactor volume 12 for receiving cell cultures within a fluid growth medium. The perfusion bioreactor 10 can be positioned in association with a rotating shaft coupled to a stirrer 13 to stir the cell cultures contained within the bioreactor volume 12.

[0074] The perfusion bioreactor 10 can be made of various materials. For example, the bioreactor 10 can be made of metal, such as stainless steel, and can be designed for reuse. Alternatively, the perfusion bioreactor 10 can comprise a single-use bioreactor made of a rigid polymer or a flexible polymer membrane. For example, when made of a rigid polymer, the bioreactor walls can be self-supporting. Alternatively, the bioreactor 10 can be made of a flexible polymer membrane or conformal material, which can be liquid-impermeable and may have an internal hydrophilic surface. In one embodiment, the perfusion bioreactor 10 can be made of a flexible polymer membrane designed to be inserted into a rigid structure, such as a metal container, to take on a desired shape.

[0075] The perfusion bioreactor 10 can have any suitable volume. For example, the volume of the perfusion bioreactor 10 can typically be greater than about 1 L, such as greater than about 5 L, such as greater than about 10 L. In embodiments, the volume of the perfusion bioreactor 10 is typically less than about 400 L, such as less than about 250 L, such as less than about 100 L. Alternatively, the perfusion bioreactor 10 can have a relatively large volume. For example, the perfusion bioreactor can have a volume greater than 250 L, such as greater than 500 L, such as greater than 750 L, such as greater than 1000 L, such as greater than 1500 L, and typically less than about 4000 L, such as less than about 3000 L, and for example, a volume from about 10 L to about 4000 L.

[0076] The perfusion bioreactor 10 may also include various other components and equipment that allow for the culture and propagation of biological cells, such as baffles, ejectors, gas supplies, heat exchangers, etc. Furthermore, the perfusion bioreactor 10 can be connected to various sensors, such as pH sensors, gas sensors, temperature sensors, etc.

[0077] The perfusion bioreactor 10 is designed to continuously receive various inputs, such as nutrient media, and continuously remove used media in order to maintain pseudo-steady-state conditions within the cell cultures contained within the bioreactor 10. For example, in one embodiment, the perfusion bioreactor 10 is operated to maintain a relatively constant volume of cell cultures and media. For example, the perfusion bioreactor 10 can be operated such that volume variations within the bioreactor do not exceed 10%, such as not exceeding about 8%, such as not exceeding about 5%, such as not exceeding about 3%.

[0078] Various methods exist for removing used media from the perfusion bioreactor 10 without consuming biological cells. For example, in one embodiment, the perfusion bioreactor may include an attachment device, such as capillary fibers or a membrane, to which cells bind, thereby preventing their release. In other embodiments, the perfusion bioreactor 10 may include a filtration device 15 that, together with the bioreactor, maintains a desired cell density. By continuously removing used media from the perfusion bioreactor 10 and replacing it with fresh media, nutrient levels can be controlled and maintained to modify growth conditions within the bioreactor. Furthermore, cellular waste can be removed in a controlled manner to avoid toxicity.

[0079] The perfusion bioreactor 10 may include multiple ports. These ports may allow supply and feed lines to enter and exit the bioreactor 10 for the addition and removal of fluids and other materials. Additionally, one or more ports may be connected to one or more probes to monitor conditions within the perfusion bioreactor 10.

[0080] For example, in Figure 1 In the illustrated embodiment, the perfusion bioreactor 10 includes an effluent port 14 and an influent port 18. The effluent port 14 is used to continuously or periodically remove liquid media from the perfusion bioreactor 10. The effluent port 14 may be in fluid communication with a pump 22 for controlling the flow rate. On the other hand, the influent port 18 may be in fluid communication with a nutrient medium supply source 16 and a pump 20. The pump 20 may be designed to pump a controlled amount of nutrient medium feed into the perfusion bioreactor 10 through the influent port 18. In one embodiment, only a single influent port 18 is required to supply nutrient medium to the perfusion bioreactor 10. However, in other embodiments, multiple ports may be used to feed nutrient medium, base medium, and / or any other components, such as pH adjusters, gases such as oxygen, nitrogen, and carbon dioxide.

[0081] As used herein, a nutrient medium or nutrient means any fluid, compound, molecule, or substance that can increase the mass of a biological product, such as any substance that an organism can use to survive, grow, or otherwise add biomass. For example, a nutrient feed may include gases used for respiration or any type of metabolism, such as oxygen or carbon dioxide. Other nutrient media may include carbohydrate sources. Carbohydrate sources include complex sugars and monosaccharides, such as glucose, maltose, fructose, galactose, and mixtures thereof. Nutrient media may also include amino acids. Amino acids may include: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid, their single stereoisomers, and racemic mixtures thereof. In some embodiments, the amino acid is glutamic acid, glutamine, lysine, tyrosine, or valine.

[0082] The nutrient medium may also contain one or more vitamins. Vitamins that may be included in the nutrient medium include B vitamins, such as B12. Other vitamins include vitamin A, vitamin E, riboflavin, thiamine, biotin, and mixtures thereof. The nutrient medium may also contain one or more fatty acids and one or more lipids. For example, the nutrient medium feed may include cholesterol, steroids, and mixtures thereof. The nutrient medium may also provide proteins and peptides to the bioreactor. Proteins and peptides include, for example, albumin, transferrin, fibronectin, fetal globulin, and mixtures thereof. The growth medium in this disclosure may also include growth factors and growth inhibitors, trace elements, inorganic salts, hydrolysis products, and mixtures thereof. Trace elements that may be included in the growth medium include trace metals. Examples of trace metals include cobalt, nickel, etc.

[0083] like Figure 1As shown, the system further includes a biomass sensor 24. The biomass sensor 24 can be used to determine the biomass concentration within the perfusion bioreactor 10. As used herein, "biomass concentration" refers to the volume contained within the cells (typically living cells) in the bioreactor, relative to the total volume (mL / mL) of the bioreactor 10, which refers to the total filling volume (liquid volume) of the bioreactor, containing liquid culture medium, cells, cell debris, etc. For example, the biomass sensor 24 can be a capacitance probe. Cells within the perfusion bioreactor 10, containing intact plasma membranes, act as capacitors under the influence of an electric field. The non-conductive nature of the plasma membrane allows charge accumulation. The resulting capacitance can then be measured. For example, the biomass sensor 24 can periodically or continuously measure the biomass within the perfusion bioreactor 10 using radio frequency impedance. For example, RF impedance allows for the measurement of the dielectric properties of the cell suspension. The obtained measurements can be used to deduce the biomass content (such as live cell volume) and other cell characteristics such as cell diameter present in the perfusion bioreactor 10 (see, for example, U.S. Patent No. 9,568,449, the disclosure of which is incorporated herein by reference in its entirety, including the disclosure of biomass content measurements). The biomass sensor 24 can be a reusable device or a single-use device. In one embodiment, for example, the biosensor can be a single-use, placement patch sensor.

[0084] When using a capacitance probe, it can operate in a frequency range from approximately 500 kHz to approximately 20,000 kHz. The capacitance measurement range is from approximately 0 to approximately 400 pF / cm. The conductivity range is from approximately 1 to approximately 40 mS / cm. Suitable methods for measuring biomass concentration using a capacitance probe are provided in the examples. Suitable capacitance probes include, for example, those from Sartorius Stedim Biotech for single-use applications. ViaMass and, for example, the Futura 12mm probe from Aber Instruments for reusable applications.

[0085] In addition to a capacitive probe, the biomass mass sensor 24 can be any other suitable instrument capable of monitoring or determining biomass concentration or cell count. For example, in an alternative embodiment, the biomass sensor 24 can be an optical cell counter. For instance, optical cell counters, commercially available under the name COUNTESS II or COUNTESS II FL Automated Cell Counter, are available from Thermo Fisher Scientific. Optical cell counters include autofocus and counter algorithms that identify cells within a population to determine biomass concentration. Methods for determining biomass concentration (biovolume) using optical cell counters are known in the art, and biomass concentration is calculated using measurements of cell counts combined with cell diameter from an optical device. Additional methods for determining biomass concentration include, for example, microscopy-based methods.

[0086] like Figure 1 As shown, the biomass sensor 24 can be contained within the volume 12 of the perfusion bioreactor 10 to maintain close contact with the cell culture contained within the bioreactor. Figure 1 In the illustrated embodiments, for example, the biomass sensor 24 may be a capacitive probe as described above.

[0087] Alternatively, the system may include at least one sample collection subsystem that obtains biomass samples from the perfusion bioreactor and analyzes the biomass concentration and / or other components of the samples. For example, in one embodiment, the perfusion bioreactor 10 may be in fluid communication with an automated sampling and testing system. Biomass samples may be fed into a sterile automated sampler to deliver the samples to a liquid handling robot, which, if necessary, automates sample preparation. The sampling and testing system can measure and monitor any parameters within the cell culture, including cell counts and biomass concentration. An example of a modular automated sampling system is sold by Lonza Ltd. under the name MAST. Automated sampling systems are described in U.S. Patent Publication Nos. 2014 / 0087413, 9,389,151, 9,322,749, 2015 / 0019140, and 2016 / 0025601, all of which are incorporated herein by reference in their entirety.

[0088] When using an automated sampling system to measure biomass concentration, the biomass sensor may include a capacitive probe or an optical cell counter.

[0089] The biomass sensor 24 can acquire readings at least every 6 hours, such as at least every 4 hours, such as at least every 2 hours, such as at least every hour, such as at least every 30 minutes, such as at least every 15 minutes, such as at least every 10 minutes. In one embodiment, the biomass sensor 24 can continuously monitor the biomass concentration within the perfusion bioreactor 10.

[0090] The biomass sensor 24 can be communicated with the controller 26 and the pump 20. For example, the controller 26 may include one or more programmable devices, such as one or more microprocessors. The controller 26 can be configured to receive biomass concentration measurements from the biomass sensor 24. Based on the information received from the biomass sensor 24, the controller 26 can be configured to control the flow rate of the nutrient medium entering the perfusion bioreactor 10 by controlling the pump 20.

[0091] In conventional perfusion bioreactors, the perfusion rate is typically kept within a relatively narrow range. However, in the process of this disclosure, in order to produce inoculum with significantly improved cell density and viable cell count, the perfusion rate, or the rate at which nutrient media are fed into the perfusion bioreactor 10, varies rapidly with the increase in biomass or cell culture within the perfusion bioreactor 10.

[0092] For example, according to this disclosure, the flow rate of the nutrient medium from the nutrient medium supply source 16 varies in a directional proportion to the current amount of biomass contained within the perfusion bioreactor 10, as determined by the biomass sensor 24. The biomass sensor 24 can perform real-time biomass concentration measurements, which are fed to a controller 26 that allows for fully automated nutrient medium flow rate through the bioreactor. During this process, for example, the nutrient medium flow rate gradually increases according to the concentration of live biomass. For example, in one embodiment, the controller 26 can be programmed with an algorithm that determines the nutrient medium flow rate based on information received from the biomass sensor 24. In one embodiment, the algorithm may be based on the following:

[0093]

[0094] Where K is the biovolume ratio feed rate, and has units of mL / % biovolume / day;

[0095] Biological volume (shown as) ) is the percentage of reactor volume (fill volume) inside the cell membrane, expressed as a%.

[0096] V is the volume of the vessel in mL; and

[0097] P is the total bioreactor filling feed rate, expressed as vessel volume per day.

[0098] This relationship can also be represented as:

[0099]

[0100] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0101] This is a biovolume fraction and refers to the volume (fill volume) of the perfused bioreactor within the cell membrane of the cell, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0102] P is the perfusion rate expressed as mL feed / mL bioreactor / day. Another way to express this, especially when using capacitors to obtain biomass, is based on the percentage of the volume within the cell membrane of living cells (VCV) divided by the total filling volume of the bioreactor (i.e., occupied by media and cells / debris, etc.).

[0103] The above relationships can be determined for any specific cell culture grown in the perfusion bioreactor 10, as well as based on various other process conditions. K in the above equations can be determined experimentally or through theoretical calculations. For example, in one embodiment, K can range from approximately 1 × 10⁻⁶. -9 Approximately 250×10 -9 Approximately 1×10 -9 Approximately 50×10 -9 or about 1×10 -9 Approximately 20×10 -9 Including approximately 1×10 -9 Approximately 10×10 -9 Approximately 4×10 -9 Approximately 9×10 -9 or approximately 7×10 -9 Inequality. As described in this paper, it has been surprisingly found that using biovolume (the percentage of reactor volume inside the cell membrane) provides a better predictor of the required nutrient medium flow rate than measurements using live cell density (VCD) (i.e., cell count). VCD assumes that the rate of nutrient consumption per cell is constant, and therefore only the number of cells matters. However, a more accurate prediction of nutrient use is based on biovolume, as described in this paper, when larger cells consume more nutrients than smaller cells.

[0104] In addition to controlling the feed rate of the nutrient medium entering the infusion bioreactor 10, such as Figure 1The processes and systems illustrated in this disclosure can also be configured to control the rate at which liquid media is drawn from bioreactor 10 using pump 22 through outlet port 14. For example, in one embodiment, the system may include a weighing device 28, such as a load sensor. The weighing device 28 can monitor the weight of the liquid media and biomass contained within the perfusion bioreactor 10. Figure 1 As shown, the weighing device 28 can be connected to the controller 26 and the pump 22. Based on information received from the weighing device 28, the controller 26 can be configured to control the amount of liquid medium drawn from the perfusion bioreactor 10 using the pump 22. In addition to relying on the information received from the weighing device 28, the controller 26 can also consider increasing the flow rate of the nutrient medium entering the bioreactor when determining the rate at which the fluid medium is drawn from the bioreactor.

[0105] In addition to the weighing device 28, the system may also include other means for determining the amount of liquid medium injected into the bioreactor 10. For example, in an alternative embodiment, the system may include a volume level indicator that monitors the volume of the liquid medium within the bioreactor 10. The volume level indicator may also be configured to communicate with the controller 26.

[0106] In one embodiment, controller 26 can be configured to operate perfusion bioreactor 10 to maintain a relatively constant volume. For example, during the process, the volume may vary by no more than about 20%, such as no more than about 15%, such as no more than about 10%, such as no more than about 5%, such as no more than about 2%.

[0107] The processes and systems disclosed herein can produce inoculum for downstream cell production with extremely high cell density and viable cell count. For example, the cell density or biomass concentration of the cell culture in the perfusion bioreactor can be increased by an amount greater than about 30 days, such as greater than about 40 days, such as greater than about 50 days, such as greater than about 60 days, such as greater than about 70 days, such as greater than about 80 days, such as greater than about 90 days, such as greater than about 100 days, such as greater than about 110 days, such as greater than about 120 days. For example, the biomass concentration can be increased by more than 150 days, such as greater than 200 days, such as greater than 250 days.

[0108] After the desired quantity has been grown in the perfusion bioreactor, the inoculum or cell culture is transferred to a larger downstream bioreactor for continued growth and harvesting of the bioproduct. The cell culture can be maintained in the perfusion bioreactor for a sufficient time to achieve the desired cell density or biomass concentration. For example, according to this disclosure, the cell culture or inoculum can have a density greater than about 10 × 10⁻⁶. 6 cells / mL, such as greater than approximately 30 × 10⁶ cells / mL 6cells / mL, such as greater than approximately 50 × 10⁶ cells / mL 6 cells / mL, such as even greater than 70 × 10⁶ cells / mL 6 A cell density of cells / mL. In this example, the cell density achieved in the perfusion bioreactor can be 100 × 10⁶ cells / mL. 6 cells / mL, 200×10 6 cells / mL, 220×10 6 cells / mL, 250×10 6 Cells / mL or higher. However, the desired cell density can depend on various process conditions and the type of cell culture produced.

[0109] Generally, any suitable inoculum can be produced using the processes and systems disclosed herein. In one embodiment, for example, the inoculum may contain mammalian cells.

[0110] Increasing the cell density and / or viable cell count of the inoculum can yield numerous benefits and advantages during downstream production. For example, inoculums with high cell density can reduce the amount of time cell cultures are held in large downstream production bioreactors. Reducing the amount of incubation time in production bioreactors directly impacts process efficiency. For instance, large-scale commercial bioreactors occupy a significant portion of the footprint in production facilities. Therefore, incubation time in production bioreactors is a limiting event for production efficiency. However, with the processes and systems of this disclosure, incubation time in downstream production bioreactors can be significantly reduced, resulting in increased space-time yield. In fact, these benefits and advantages can be achieved even with increased inoculum incubation time in perfusion bioreactors.

[0111] refer to Figure 2 The figure illustrates one embodiment of a production process for producing biological products. As shown, in one embodiment, inoculum 40 may first be fed into a small cell culture vessel 50. For example, the cell culture vessel 50 may be a shake flask expansion device. The inoculum can be grown to a limited extent in these specialized incubator vessels. For example, the cell culture vessel 50 may typically have a volume greater than about 0.5 L, such as greater than about 1 L, such as greater than about 2 L, and typically less than about 5 L, such as less than about 4 L, such as less than about 3 L.

[0112] The inoculum is then fed from cell culture dish 50 into the perfusion bioreactor 10 of this disclosure. For example... Figure 2As shown, the perfusion bioreactor 10 can be a shaking bioreactor, a wave-shaped bioreactor, or a stirred tank bioreactor. In one embodiment, the perfusion bioreactor 10 can have a volume of about 5L to about 4000L, such as about 10L to about 3000L, such as about 50L to about 2000L. The perfusion bioreactor 10 can be a stainless steel vessel or a disposable bag-type bioreactor that can be used as a liner in the vessel.

[0113] The incubation time within the perfusion bioreactor 10 can vary depending on the inoculum produced and the desired final cell density. For example, the incubation time of the inoculum with the perfusion bioreactor 10 can typically be greater than about 3 days, such as greater than about 5 days, such as greater than about 7 days, such as greater than about 9 days, and typically less than about 15 days, such as less than about 12 days, such as less than about 11 days. As described above, the perfusion bioreactor 10 is particularly suitable for producing inoculum with significantly improved cell density and viable cell count.

[0114] The inoculum is then fed from the perfusion bioreactor 10 into the production bioreactor 60 to produce the bioproduct. In one embodiment, the production bioreactor 60 may have a volume greater than about 500 L, such as greater than about 600 L, such as greater than about 700 L, and typically less than about 30,000 L, such as less than about 20,000 L, such as less than about 10,000 L. Typically, the production bioreactor 60 has a larger volume than the perfusion bioreactor 10. For example, the volume ratio between the perfusion bioreactor and the production bioreactor may be from about 1:3 to 1:40, such as from about 1:4 to about 1:10 (e.g., a 4,000 L perfusion bioreactor and a 20,000 L production (e.g., batch-fed) bioreactor).

[0115] Due to the increased cell density of the inoculum fed into the production bioreactor 60, the incubation time within the bioreactor 60 can be significantly reduced. For example, conventional systems typically require an incubation time of 15 days or longer. However, the incubation time within the production bioreactor 60 according to this disclosure can be less than about 13 days, such as less than about 12 days, such as less than about 11 days, such as less than about 10 days, such as less than about 9 days, such as even less than about 8 days. Incubation times are typically greater than about 3 days, such as greater than about 5 days. Any reduction in the incubation time within the production bioreactor 60 has a significant impact on improving the efficiency of the overall process.

[0116] By producing inoculum with higher cell density and eliminating non-productive start-up days in the production bioreactor, higher titers can be achieved in a shorter timeframe. To illustrate this effect, Figure 3 This is an illustration of some of the benefits of the process disclosed herein. Figure 3The graphs in the figure depict the changes in live cell density and titer over time in the production bioreactor. Sample 1 represents a routine process, where the inoculum fed into the production bioreactor has a concentration of 5 × 10⁻⁶ cells / mL. 5 A cell density of cells / mL. However, the second sample prepared according to this disclosure represents a cell density of 10 × 10⁻⁶ cells / mL. 6 A cell density of 100 cells / mL was used to feed the inoculum into the production bioreactor. As shown in the figure, the second sample produced a significantly better growth rate and a higher overall titer. For example, after 15 days, the first sample reached a titer of 4.95 g / L, while the second sample reached a titer of 6.13 g / L in just 12 days. The titer was divided by the incubation time to obtain the space-time yield. The first sample produced a space-time yield of 0.3 g / L / day, while the second sample produced a space-time yield of 0.51 g / L / day. Therefore, the space-time yield produced by the process according to this disclosure is increased by 50%.

[0117] As described above, the perfusion bioreactor of this disclosure can produce a significant and dramatic increase in cell density compared to many conventional processes. In fact, through experimental procedures, the perfusion bioreactor of this disclosure can produce cells with a density of 80 × 10⁻⁶. 6 Cells / mL, such as 90 × 10⁻⁶ 6 Cell cultures with a cell density of 100 × 10⁶ cells / mL. In fact, the perfusion bioreactor of this disclosure has been shown to produce 100 × 10⁶ cells / mL. 6 Cell density of 1 cell / mL or greater.

[0118] Due to the increased cell density, the perfusion bioreactor of this disclosure can be used to produce final products in addition to producing inoculum. For example, in one embodiment, the perfusion bioreactor of this disclosure can be used to incubate cell cultures, and the bioproduct can be harvested directly from the cell cultures. In one embodiment, the cell cultures produced in the perfusion bioreactor can be fed into a downstream purification process for harvesting the bioproduct. Therefore, the present invention also relates to a cell culture production process comprising:

[0119] Inoculum comprising host cells expressing biological products is produced by the method of the first aspect of the present invention;

[0120] Inoculation materials are introduced into production bioreactors, and cell cultures are introduced into perfusion bioreactors;

[0121] Culture host cells to produce biological products;

[0122] Harvesting biological products from cell cultures; and

[0123] Optionally, one or more purification steps may be performed on the biological product.

[0124] In one embodiment, the inoculum is introduced into the production bioreactor until the final density is at least 5 × 10⁻⁶. 6 cells / mL, such as at least 8 or 10 × 10⁶ cells / mL 6 Cells / mL. For example, this could represent a 4 to 10-fold dilution of inoculum from the N-1 process of this invention.

[0125] In the embodiments, cells express or produce products, such as recombinant therapeutic or diagnostic products. Examples of cell-generated products include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimics (peptide molecules that specifically bind to antigens but are not structurally related to antibodies, such as DARPins, affibodies, adnectins, or IgNARs), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutics (e.g., anticancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cell therapeutics (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines, or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics, or amino acids. In the embodiments, the apparatus, facilities, and methods can be used to produce biosimilars.

[0126] As mentioned, in the embodiments, the apparatus, facilities, and methods allow for the production of eukaryotic cells, such as mammalian cells or lower eukaryotic cells, such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as Gram-positive or Gram-negative cells, and / or products of eukaryotic or prokaryotic cells, such as proteins, peptides, antibiotics, amino acids, and nucleic acids (such as DNA or RNA), synthesized by eukaryotic cells on a large scale. Unless otherwise stated herein, the apparatus, facilities, and methods may include any desired volume or production capacity, including but not limited to laboratory-scale, pilot-scale, and full-scale production capacity.

[0127] Furthermore, and unless otherwise stated herein, apparatus, facilities, and methods may include any suitable reactor, including but not limited to stirred tanks, airlift reactors, fiber, microfiber, hollow fiber, ceramic-based, fluidized bed, fixed bed, and / or sputtered bed bioreactors. As used herein, a “reactor” may comprise a fermenter or fermentation unit or any other reaction vessel, and the terms “reactor” and “fermenter” are used interchangeably. For example, in some aspects, exemplary bioreactor units may perform one or more or all of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flows of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Example reactor units, such as fermentation units, may contain multiple reactors within a unit. For example, the unit may have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit. A facility may contain multiple units having one or more reactors within the facility. In various embodiments, the bioreactor may be suitable for batch, semi-feed batch, fed batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor may have a volume between about 100 mL and about 50,000 L. Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 L, 2 L, 3 L, 4 L, 5 L, 6 L, 7 L, 8 L, 9 L, 10 L, 15 L, 20 L, 25 L, 30 L, 40 L, 50 L, 60 L, 70 L, 80 L, 90 L, 100 L, 150 L, 200 L, 250 L, 300 L, 350 L, 400 L, 450 L, 500 L, 550 L, 600 L, and 65 L. 0 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, 30,000 liters, 40,000 liters, and / or 50,000 liters. Furthermore, suitable reactors can be reusable, single-use, disposable, or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and / or glass.

[0128] Once the biosynthesis of the product by the producing cells has progressed to a satisfactory point, the product can be harvested, for example, by removing the culture medium and separating the supernatant from the cells and cell debris. The product can be subjected to one or more purification / treatment steps, such as affinity chromatography, ion exchange chromatography, filtration, and / or virus inactivation, to obtain a purified product. The product can also be combined with one or more pharmaceutically acceptable carriers, excipients, or diluents to produce compositions, such as formulations containing one or more of buffers, surfactants, stabilizers (e.g., trehalose, sucrose, glycerol), amino acids (e.g., glycine, histidine, arginine), metal ions / chelating agents, salts, and / or preservatives.

[0129] The apparatus, devices, and methods described herein are suitable for culturing any desired cell lines, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the apparatus, devices, and methods are suitable for culturing suspension cells or adherent (adhesive) cells and are suitable for being configured for the production of pharmaceutical and biopharmaceutical products—such as peptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses, such as those for cell and / or viral therapies. In one embodiment, the host cell is a mammalian cell. Exemplary species from which host cells can be derived include humans, mice, rats, Chinese hamsters, Syrian hamsters, monkeys, apes, dogs, horses, ferrets, and cats. In an embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the host cell is a CHO-K1 cell. Cells, DG44CHO cells, DUXB11 CHO cells, CHO-S cells, CHO-GS knockout cells (CHO cells in which all endogenous copies of the glutathione synthase (GS) gene have been inactivated), FUT8 knockout cells, CHOZN cells, or CHO-derived cells. CHO GS knockout cells (e.g., GS-KO cells) are, for example, FUT8 knockout cells, CHOZN cells, or CHO-derived cells. GS knockout cells (e.g., GS knockout cells) Cell-CHOK1SV Lonza Biologics, Inc. CHO FUT8 knockout cells are, for example... FUT8 knockout cells (Lonza Biopharmaceuticals).

[0130] In the embodiments and unless otherwise stated herein, the apparatus, facilities, and methods described herein may also include any suitable unit operations and / or equipment not otherwise mentioned, such as operations and / or equipment for separating, purifying, and isolating such products. Any suitable facility and environment may be used, such as conventional modular construction facilities, mobile and temporary facilities, or any other suitable building, facility, and / or layout. For example, in some embodiments, a modular cleanroom may be used. Additionally, unless otherwise stated, the apparatus, systems, and methods described herein may be housed in and / or performed in a single location or facility, or alternatively housed in and / or performed in one or more locations and / or facilities.

[0131] Example

[0132] Example 1: Biovolume-based perfusion reactor feed predicts positive culture performance and outperforms cell count-based feed.

[0133] Capacitance measurement of biomass concentration (biovolume)

[0134] Culture biomass concentration (biovolume) was measured using an Aber Futura capacitance probe (Aber Instruments Ltd, Aberystwyth, UK). Capacitance was measured every 30 seconds at 1000 kHz. The capacitance signal was filtered using a 30-sample moving average filter, and no electrode polarization was applied to the signal. In calibration experiments, capacitance values ​​were correlated with biovolume (biomass concentration), where daily samples were extracted from growing bioreactor cultures at different live cell concentrations. The live cell concentration and mean cell diameter for each sample were determined using a Nova Bioprofile Flex (Nova Biomedical, Waltham, MA). The live biovolume was determined from the aforementioned measurements using the Flex by assuming a spherical cell geometry.

[0135]

[0136] in VCC is the volume fraction of live organisms in mL / mL, VCC is the concentration of live cells in cells / mL, and D is the average cell diameter in μm.

[0137] The calibration experiment yielded It has a linear correlation with capacitance.

[0138] φ=m*C+b

[0139] in The volume of living organisms is expressed in mL / mL, m is the slope of the calibration curve (mL*cm / (mL*pF)), C is the capacitance (pF / cm), and b is the intercept of the calibration curve (mL / mL).

[0140] Special note regarding biocapacity: Biocapacity fraction, which is analogous to concentration (mL / mL), is an analogue to VCC (cells / mL). Live biocapacity (mL) is the total biocapacity and is an analogue to total cell count (cells). In other words, both biocapacity fraction and VCC are concentrations, and live biocapacity and total cell count are the sum of the entire volume of the reactor.

[0141] Perfusion culture operation for experiments in feed handling space

[0142] Perfusion culture was performed using a single CHO cell line expressing a monoclonal antibody. The culture was inoculated at 0.5 × 10⁻⁶ cells / year. 6 Cells were seeded at a viable cell concentration of 100 cells / mL into a chemically defined basal medium (basal medium + 1.9 vol% SF102 (concentrated nutrient feed)) at pH 6.9. Dissolved oxygen was maintained at >= 40% air saturation. Cells were allowed to expand with supplemental feed until day 6, at which point perfusion was initiated at 1 vessel volume (vv) / day (perfusion medium – basal medium + 4.21 vol% SF102). As cell expansion continued, the perfusion rate was increased to a maximum of 2 vv / day. Once the culture achieved the desired cell concentration (or biovolume fraction, depending on the control strategy for this particular operation), cell exudation was initiated via capacitively controlled exudation to maintain the culture at a constant cell concentration or biovolume fraction. Again, the perfusion feed rate was manually adjusted according to the experiment to achieve the desired cell-to-perfusion rate or biovolume-to-perfusion rate.

[0143] Procedure for perfusing N-1 culture

[0144] The perfused N-1 culture was injected with 0.5 × 10⁻⁶ mg / L. 6 Cells were inoculated at a viable cell concentration of [number] cells / mL into a chemically defined basal medium. Cells were allowed to expand until day 4 of perfusion initiation. The perfusion feed rate was determined by the biocapacity fraction, as predicted from capacitance readings, as follows:

[0145]

[0146] Where P is the infusion feed rate in vv / day, K is the biovolume ratio infusion rate (mL feed / mL biovolume / day), and It is the biomass volume (mL biomass volume / mL bioreactor).

[0147] Cell division is allowed, and the perfusion rate is controlled accordingly up to 100 × 10⁻⁶. 6 Cells / mL. Figures 4A to 4B An illustrative example of this variable feed is shown in the figure, used for three CHO cell clones (C1 to C3).

[0148] Creation of the feeding operation space

[0149] For perfusion medium feeding, two feeding considerations were explored: supplementing sufficient nutrients to meet cell needs without overfeeding, and removing waste products from the culture through dilution. The perfusion medium was divided into two components: basal medium and concentrated nutrient supplement. Different nutrient richness levels of perfusion medium were obtained by varying the amount of nutrient supplement (SF102) added to the basal medium. More nutrient supplement resulted in a richer perfusion medium.

[0150] Perfusion media were prepared by adding varying amounts of nutrient supplements. The cell-to-perfusion rate or biovolume-to-perfusion rate for each culture medium composition was then varied, and the steady-state behavior of each culture was observed. Cultures resulting in stable homeostasis for at least 5 days were considered acceptable performance, while cultures resulting in reduced viability or apoptosis (and typically culture collapse) were considered unacceptable performance.

[0151] For each perfusion culture condition, the cell-to-perfusion rate or biovolume-to-perfusion rate was plotted against the cell-to-concentrated nutrient feed rate or biovolume-to-concentrated nutrient feed rate. The biovolume-to-concentrated nutrient feed replenishment rate or cell-to-concentrated nutrient feed replenishment rate was determined as the biovolume-to-perfusion rate or cell-to-perfusion rate multiplied by the amount of concentrated nutrient feed added to the basal perfusion medium for that particular medium (expressed as a volume fraction).

[0152] Implementation plan for feed operation space learning in perfused N-1 cultures

[0153] Once the operating space is established, it specifies the range of perfusion rates and concentrated nutrient feed replenishment rates that result in acceptable (and unacceptable) culture performance (see [link to operating space]). Figure 5 The upper and lower dashed lines (indicated by the operating space) specify the perfusion feed conditions that result in acceptable culture performance in non-steady-state or N-1 amplification cultures. To achieve this, conditions are selected near the middle of the predetermined feed operating space (see [reference]). Figure 5 (The rhombus in the middle).

[0154] result

[0155] At the edge of acceptable culture performance, a set of initial perfusion cultures at different nutrient replenishment rates supported the hypothesis of constant nutrient consumption per cell, where unstable cultures were obtained at excessively high and low SF102 nutrient feed / cell rates (see X-marked). Figure 6 ).

[0156] Based on initial observations of the proposed high and low nutrient feed rate limits per cell, and the assumption that the limits would be constant, more aggressive culture conditions were attempted, which predicted the use of less medium (lower CSPR) but still operation within the optimal window for nutrient feed per cell (+ sign). Figure 6 Surprisingly, unstable cultures and signs of nutrient overfeeding, including changes in cell size, were observed under the new (+) conditions.

[0157] The assumption of a constant nutrient requirement per cell is inaccurate, and instead, the required nutrients should be predicted on a per-biovolume basis. This explains the fact that larger cells require more nutrients due to their greater cellular mechanisms, and conversely, smaller cells require less. When the same perfusion conditions are observed on a per-biovolume basis, the selected perfusion condition (+) is actually predicted to be more correlated with the observed values ​​on the overfeeding side (circled + sign). Figure 7 Located at the top dashed line, this represents the upper limit of the acceptable range of culture performance.

[0158] An updated interpretation of the feed constraint based on the assumption of a constant per-biovolume ratio is selected, choosing new conditions within the predicted ideal feed range of the biovolume ratio feed space. Figure 7 (The triangle in the image). These conditions produce high-performance perfusion cultures.

[0159] However, as a function of CSPR, using the assumption of a constant cell-to-nutrient feed rate, this success condition is predicted to be underfeeding. Figure 8 The circled triangle is below the lower dashed line and therefore outside the “acceptable range of culture performance”.

[0160] Based on this unexpected discovery, a feeding strategy based on a constant per-biovolume was implemented (see [link]). Figure 9 As indicated, if cells are grown in an environment that is "too rich in nutrients," they cannot demonstrate acceptable growth characteristics (top image). However, cells grown within the "acceptable range of culture performance" exhibit the desired cellular characteristics of appropriate cell shape and density.

[0161] Optimize the feeding operation space in the N-1 injection process.

[0162] The effect of the range of biovolume ratio perfusion feed rates on culture performance was evaluated. N-1 cultures were perfused at four biovolume ratio perfusion rates: 5, 6.2, 7, and 8.4 mL / mL / day. These conditions were selected such that, for a single perfusion medium composition, intermediate conditions were predicted to provide good culture performance, and flanking conditions were predicted to approach the edge of the acceptable operating space, but still within the boundaries (see [link to relevant documentation]). Figure 10 ).

[0163] As predicted by the operating space, each culture successfully achieved a cell density of at least 75 x 10^6 cells / mL. However, conditions closer to the edge of the space resulted in slower growth, and correspondingly, higher media consumption (8.4 and 5 BVSPR) compared to those at the center of the space (and 6.2 BVSPR) (see [link to operating space description]). Figure 11A ). Figure 11B The cell density (VCC) of each of the selected conditions for CHO cell clone L1 is shown.

[0164] Finally, Figure 11B The optimal conditions specified for the single cell clone (7 mL / mL / day) (C1) were applied to four other clones (C2 to L5), all of which showed favorable growth characteristics. This supports the use of a biovolume ratio feed method as a platform approach and is not limited to just one cell line. Figure 12 ).

[0165] In summary, it was surprising to find that using a model based on the percentage of biovolume—the reactor volume (i.e., liquid volume: liquid medium, cells, cell debris, etc.) within the cell membrane—more accurately predicted the feed conditions required to deliver the desired perfusion culture.

[0166] The process of scaling up proportionally

[0167] Figure 13 This demonstrates the successful scaling-up of the N-1 process from benchtop scale to a 50 L single-use stirred tank reactor (pilot-scale). Achieving 50 × 10⁻⁶ ppm was accomplished within approximately the same incubation duration (about 9 days). 6 The target of 10 million cells / mL VCC was exceeded, and a VCC of 70 million cells / mL was achieved on day 11. These results demonstrate that automated processes can be adapted to different scales and reactor configurations.

[0168] Exemplary Examples

[0169] Example 1 is a process for producing inoculum for subsequent cell culture production, comprising: introducing cell culture into a perfusion bioreactor; feeding nutrient medium into the perfusion bioreactor at a given flow rate and removing fluid medium from the perfusion bioreactor; determining the biomass concentration over time within the perfusion bioreactor using a biomass sensor, such as a capacitive sensor, which is connected to a controller; and adjusting the flow rate of the nutrient medium entering the perfusion bioreactor based on the biomass concentration sensed by the biomass sensor, wherein the controller is configured to adjust the flow rate based on information received from the biomass sensor, wherein the nutrient medium flow rate is adjusted based on the following relationship:

[0170] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0171] This is a biovolume fraction and refers to the volume of a perfusion bioreactor within the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0172] P is the infusion rate expressed as mL feed / mL bioreactor / day.

[0173] Example 2 includes the process defined in Example 1, wherein the controller is configured to increase the flow rate of the nutrient medium as the biomass concentration increases.

[0174] Example 3 includes the process defined in any of the foregoing examples, further comprising the steps of: determining the amount of fluid medium in the perfusion bioreactor, and based on that amount, selectively increasing or decreasing the rate at which the fluid medium is extracted from the perfusion bioreactor.

[0175] Example 4 includes the process defined in Example 3, wherein the amount of fluid medium within the perfusion bioreactor is determined by weighing the perfusion bioreactor using a weighing device.

[0176] Example 5 includes the process defined in Example 4, wherein the weighing device is connected to the controller, and based on the weight information from the weighing device, the controller is configured to control the pumping device in fluid communication with the perfusion bioreactor to selectively increase or decrease the rate of extraction of the fluid medium.

[0177] Example 6 includes the process defined in Example 5, wherein the fluid medium extracted from the perfusion bioreactor is filtered to prevent biomass from being extracted from the bioreactor along with the fluid medium.

[0178] Example 7 includes the process defined in Example 3, wherein the amount of fluid medium in the perfusion bioreactor is determined by measuring the volume.

[0179] Example 8 includes the process defined in any of the foregoing examples, wherein the cell culture has a cell density, and the cell density increases over time in the perfusion bioreactor.

[0180] Example 9 includes the process defined in Example 8, wherein the volume of the fluid medium and cell culture remains constant during the process.

[0181] Example 10 includes the process defined in any of the foregoing examples, wherein the cell culture comprises mammalian cells.

[0182] Example 11 includes the process defined in any of the foregoing examples, wherein the biomass sensor determines the biomass concentration in the injection reactor at least every 6 hours.

[0183] Example 12 includes the process defined in any of the preceding examples, wherein the perfusion bioreactor has a volume of about 10 liters to about 4000 L.

[0184] Example 13 includes the process defined in any of the preceding examples, wherein after the incubation period, the process further includes transferring the cell culture from the perfusion bioreactor to a second bioreactor having a volume larger than that of the perfusion bioreactor, the volume ratio between the perfusion bioreactor and the second bioreactor being 1:3 to 1:40, such as about 1:4 to about 1:10.

[0185] Example 14 includes the process defined in Example 13, wherein the cell culture continues to grow in a second bioreactor in a batch feeding manner.

[0186] Example 15 includes the process defined in Example 13 or 14, wherein the cell culture is maintained in a perfusion bioreactor for about 3 days to about 12 days and in a second bioreactor for less than about 12 days, such as less than about 10 days.

[0187] Example 16 includes the process defined in any of the foregoing examples, wherein the cell culture reaches a density greater than about 10 × 10⁻⁶ within a perfusion bioreactor. 6 cells / mL, such as greater than approximately 30 × 10⁶ cells / mL 6 cells / mL, such as greater than approximately 50 × 10⁶ cells / mL 6 cells / mL, such as greater than approximately 70 × 10⁻⁶ cells / mL 6 Cell density of cells per mL.

[0188] Example 17 includes the process defined in any of the foregoing examples, wherein the cell culture reaches 100 × 10⁻⁶ cells / mL in a perfusion bioreactor. 6 Cell density of 1 cell / mL or greater.

[0189] Example 18 includes the process defined in any of the preceding examples, wherein the cell culture has a cell density, and wherein the cell density increases by at least 60% per day within the perfusion bioreactor.

[0190] Example 19 is a system for producing inoculum for subsequent cell culture production processes, comprising: a perfusion bioreactor; a nutrient medium feed in fluid communication with the perfusion bioreactor for feeding nutrient medium into the perfusion bioreactor to induce cell culture growth; an effluent for removing fluid medium from the perfusion bioreactor; a pumping device in fluid communication with the effluent from the perfusion bioreactor to remove a controlled amount of fluid medium from the perfusion bioreactor; a weighing device for monitoring the weight of the perfusion bioreactor; a biomass sensor, such as a capacitive sensor, in fluid communication with the perfusion bioreactor to determine the biomass concentration within the perfusion bioreactor; and a controller in communication with the biomass sensor and the weighing device, the controller being configured to control the nutrient medium feed to increase or decrease the flow rate of the nutrient medium fed into the perfusion bioreactor based on information received from the biomass sensor, the controller also being configured to control the pumping device to increase or decrease the flow rate of fluid medium extracted from the perfusion bioreactor based on information received from the weighing device, wherein the controller controls the flow rate of the nutrient medium entering the perfusion bioreactor based on the following relationship:

[0191] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0192] This is a biovolume fraction and refers to the volume of a perfusion bioreactor within the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0193] P is the infusion rate expressed as mL feed / mL bioreactor / day.

[0194] Example 20 is a system as defined in Example 19, wherein the controller includes one or more microprocessors.

[0195] Example 21 is a system as defined in any one of Examples 19 to 20, wherein the perfusion bioreactor has a volume from about 10 liters to about 250 liters.

[0196] Example 22 is a system as defined in any one of Examples 19 to 21, further comprising a second bioreactor in fluid communication with the perfusion bioreactor, the second bioreactor being configured to receive cell cultures from the perfusion bioreactor, the second bioreactor having a volume larger than that of the perfusion bioreactor, the volume ratio between the perfusion bioreactor and the second bioreactor being from 1:3 to 1:40, such as from about 1:4 to about 1:10.

[0197] Example 23 is a cell culture production process comprising: introducing cell culture into a perfusion bioreactor; feeding a nutrient medium into the perfusion bioreactor at a given flow rate and removing a fluid medium from the perfusion bioreactor; determining the biomass concentration over time within the perfusion bioreactor using a biomass sensor connected to a controller; and adjusting the flow rate of the nutrient medium entering the perfusion bioreactor based on the biomass concentration sensed by the biomass sensor, wherein the controller is configured to adjust the flow rate based on information received from the biomass sensor, wherein the nutrient medium flow rate is adjusted based on the following relationship:

[0198] Where K is the bio-volume ratio perfusion rate (mL feed / mL bio-volume / day);

[0199] This is a biovolume fraction and refers to the volume of a perfusion bioreactor within the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and

[0200] P is the infusion rate expressed as mL feed / mL bioreactor / day.

[0201] Example 24 includes the process defined in Example 23, wherein the cell culture is fed into the purification process after the incubation period.

[0202] Example 25 includes the process defined in Example 23, wherein, after an incubation period, a biological product is harvested from the cell culture.

[0203] Example 26 includes a cell culture production process comprising: producing an inoculum comprising host cells expressing a biological product by any one of the methods of Examples 1 to 18;

[0204] Introduce the inoculum into the production bioreactor;

[0205] Culture host cells to produce biological products;

[0206] Harvesting biological products from cell cultures; and

[0207] Optionally, one or more purification steps may be performed on the biological product.

[0208] These and other modifications and variations to the invention can be made by those skilled in the art without departing from the spirit and scope of the invention, which is more specifically set forth in the appended claims. Furthermore, it should be understood that aspects of the various embodiments can be interchanged, in whole or in part. Moreover, those skilled in the art will understand that the foregoing description is merely exemplary and is not intended to limit the invention further described in the appended claims.

Claims

1. A process for producing an inoculum for subsequent cell culture production processes, comprising: Introduce cell cultures into a perfusion bioreactor; Nutrient media are fed into the perfusion bioreactor at a certain flow rate and fluid media are removed from the perfusion bioreactor; A biomass sensor, connected to a controller, is used to determine the biovolume fraction over time within the perfusion bioreactor; wherein the biovolume fraction is the volume of the perfusion bioreactor within the cell membrane of a cell, expressed as a percentage or fraction (mL biovolume / mL bioreactor); and The biomass sensor is calibrated to measure the biomass volume fraction; as well as The controller is configured to adjust the flow rate of the nutrient medium entering the perfusion bioreactor based on the biovolume fraction sensed by the biomass sensor. The flow rate of the nutrient medium is adjusted based on the following relationship: P=K*φ Where K is the biovolume ratio perfusion rate expressed in mL feed / mL biovolume / day; φ is the biomass volume fraction sensed by the biomass sensor; and P is the infusion rate expressed as mL feed / mL bioreactor / day.

2. The process according to claim 1, wherein the biomass sensor is a capacitive sensor.

3. The process according to claim 1 or 2, wherein the controller is configured to increase the flow rate of the nutrient medium as the biovolume fraction increases.

4. The process according to claim 1 or 2, further comprising the steps of: determining the amount of fluid medium within the perfusion bioreactor, and selectively increasing or decreasing the rate at which the fluid medium is extracted from the perfusion bioreactor based on the amount.

5. The process of claim 4, wherein the amount of the fluid medium within the perfusion bioreactor is determined by weighing the perfusion bioreactor using a weighing device.

6. The process of claim 5, wherein the weighing device is in communication with the controller, and based on weight information from the weighing device, the controller is configured to control a pumping device in fluid communication with the perfusion bioreactor to selectively increase or decrease the rate of extraction of the fluid medium.

7. The process of claim 4, wherein the amount of the fluid medium within the perfusion bioreactor is determined by measuring the volume.

8. The process according to claim 1 or 2, wherein the cell density in the cell culture in the perfusion bioreactor increases over time.

9. The process according to claim 1 or 2, wherein the cell density in the cell culture in the perfusion bioreactor increases by at least 60% per day.

10. The process according to claim 1 or 2, wherein the biomass sensor determines the biovolume fraction within the perfusion bioreactor at least every 6 hours.

11. The process according to claim 1 or 2, wherein the perfusion bioreactor has a volume of 10 L to 4000 L.

12. The process according to claim 1 or 2, wherein after the incubation period, the process further comprises transferring the cell culture from the perfusion bioreactor to a second bioreactor having a volume larger than that of the perfusion bioreactor, the volume ratio between the perfusion bioreactor and the second bioreactor being 1:3 to 1:

40.

13. The process of claim 12, wherein the volume ratio between the perfusion bioreactor and the second bioreactor is 1:4 to 1:

10.

14. The process of claim 12, wherein the cell culture continues to grow in the second bioreactor in a batch feeding manner.

15. The process of claim 12, wherein the cell culture is maintained in the perfusion bioreactor for 3 to 12 days and in the second bioreactor for less than 12 days.

16. The process of claim 12, wherein the cell culture is maintained in the perfusion bioreactor for 3 to 12 days and in the second bioreactor for less than 10 days.

17. The process according to claim 1 or 2, wherein the cell culture reaches a concentration greater than 10 × 10⁻⁶ within the perfusion bioreactor. 6 Cell density of cells per mL.

18. The process according to claim 1 or 2, wherein the cell culture reaches a concentration greater than 30 × 10⁻⁶ within the perfusion bioreactor. 6 Cell density of cells per mL.

19. The process according to claim 1 or 2, wherein the cell culture reaches a concentration greater than 50 × 10⁻⁶ within the perfusion bioreactor. 6 Cell density of cells per mL.

20. The process according to claim 1 or 2, wherein the cell culture reaches a concentration greater than 70 × 10⁻⁶ within the perfusion bioreactor. 6 Cell density of cells per mL.

21. The process according to claim 1 or 2, wherein the cell culture reaches 100 × 10⁻⁶ within the perfusion bioreactor. 6 Cell density of 1 cell / mL or greater.

22. The process according to claim 1 or 2, wherein the cell culture comprises mammalian cells.

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