Process and system for producing inoculum

Abstract

To provide an improved process for producing an inoculum of a microorganism for later transfer to a larger production bioreactor.SOLUTION: A process for producing an inoculum for a subsequent cell culture production process comprises: introducing a cell culture into a perfusion bioreactor; feeding a nutrient medium at a flow rate to the perfusion bioreactor and withdrawing a fluid medium from the perfusion bioreactor; determining biomass concentrations over time within the perfusion bioreactor using a biomass sensor, the biomass sensor being in communication with a controller; and adjusting the nutrient medium flow rate into the perfusion bioreactor based on biomass concentrations sensed by the biomass sensor, the controller being configured to adjust the medium flow rate based on information received from the biomass sensor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [Background technology] Bioreactors are devices capable of conducting biological reactions or processes on a laboratory or industrial scale and are widely used in the biopharmaceutical industry. Bioreactors can be used to produce any type of bioproduct. Bioproducts can include, for example, cell cultures and cell culture-derived materials such as beverages, biofuels, bioenergy, biochemicals, antibiotics, amino acids, enzymes, monoclonal antibodies, monomers, proteins, food cultures, biopolymers, alcohol, flavors, fragrances, and the like. In some embodiments, cell cultures can be grown for cell therapy. Cell therapy is the prevention, treatment, cure, or alleviation of human disease or injury through the administration of ex vivo engineered or modified autologous, allogeneic, or xenogeneic cells. One goal of cell therapy is to repair, replace, or restore damaged tissues or organs.

[0002] Cell cultures are typically grown in batch processes in which the biological material remains in the bioreactor until the end of the reaction time. In some of these processes, the fluid medium contained within the bioreactor can be periodically or continuously removed and re-fed to replenish nutrients contained within the fluid medium and, in some cases, to remove harmful by-products produced during the process.

[0003] Before growing a cell culture in a batch reactor as described above, an inoculation process is first performed. For example, a microbial inoculum is required to provide a population with a desired number of viable microbial cells, making it suitable for scaling up to levels suitable for commercial-scale production. Currently, the inoculation process is performed in batch mode. Nutrient limitations and the accumulation of inhibitory metabolites limit the maximum cell density that can be achieved in these conventional processes. Low cell densities during the inoculation process can result in long turnaround times in commercial batch reactors. For example, an inoculum fed into a batch reactor with a lower cell density initially requires a downstream production bioreactor that spends a significant amount of time generating cell mass instead of producing the desired bioproduct, such as a protein. Ultimately, longer incubation times are required in larger commercial batch reactors, directly impacting the efficiency of the overall process.

[0004] In view of the above, there is a need for improved methods and systems for producing a microbial inoculum for subsequent transfer to a larger production bioreactor. There is also a need for methods and systems that generate an inoculum that results in a higher viable cell count and allows for greater cell densities during the inoculation process. There is also a need for an inoculation process that produces an inoculum that can be fed to a production bioreactor and reduces the time required in the production bioreactor to produce a biological product.

[0005] [Summary of the Invention] [Problem to be solved by the invention] Generally, the present disclosure relates to an inoculation process that produces rapidly increasing cell densities and viable cell counts over the course of a culture. The inoculation process and system of the present disclosure generally involve a perfusion bioreactor in which the perfusion rate does not remain constant, but rather evolves and increases as the cell culture increases in biomass. Indeed, automated methods can be used to adjust the nutrient feed rate to the perfusion bioreactor based on real-time biomass measurements. Through this process, an inoculum can be produced with dramatically increased cell densities and / or viable cell counts. The inoculum can then be fed to a larger production bioreactor for producing bioproducts such as proteins. Through the inoculation process and system of the present disclosure, the time required for the production bioreactor can be significantly reduced, thereby dramatically increasing process throughput. It has been found that perfusion rate control is improved when based on viable cell volume / biovolume measurements, as opposed to previous methods based on viable cell density.

[0006] [Means for solving the problem] Thus, in a first aspect, the present disclosure provides a process for producing an inoculum for a subsequent cell culture production process, comprising: introducing the cell culture into a perfusion bioreactor; supplying a nutrient medium to the perfusion bioreactor at a flow rate and removing a fluid medium from the perfusion bioreactor; determining a biomass concentration over time in the perfusion bioreactor using a biomass sensor, the biomass sensor being in communication with the controller; adjusting a nutrient medium flow rate to the perfusion bioreactor based on a biomass concentration sensed by the biomass sensor, wherein the controller is configured to adjust the medium flow rate based on information received from the biomass sensor; The nutrient medium flow rate is related to the following: P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); P is the perfusion rate expressed in m feed / m bioreactor / day), adjusted based on the process.

[0007] In a related embodiment, the present invention also provides a process for producing an inoculum for a subsequent cell culture production process, comprising: introducing the cell culture into a perfusion bioreactor; supplying a nutrient medium to the perfusion bioreactor at a flow rate and removing a fluid medium from the perfusion bioreactor; determining a biovolume fraction over time in the perfusion bioreactor using a biomass sensor, the biomass sensor being in communication with the controller; adjusting a nutrient medium flow rate to the perfusion bioreactor based on the biovolume fraction, wherein the controller is configured to adjust the medium flow rate based on information received from the biomass sensor; For processes where the nutrient medium flow rate is adjusted as described above based on the following relationship: P=K*φ as described above.

[0008] This process can be used to produce an inoculum for a subsequent cell culture production process, such as the production of a desired bioproduct. The process can include introducing the cell culture into a perfusion bioreactor. A nutrient medium can be supplied to the perfusion bioreactor at a flow rate. Simultaneously, a fluid medium can be removed from the perfusion bioreactor. The fluid medium removed from the perfusion bioreactor can also be filtered to prevent cell loss. During growth of the cell culture in the perfusion bioreactor, a biomass concentration is determined over time using a biomass sensor in fluid communication with the cell culture in the perfusion bioreactor. The biomass sensor can also be in communication with a controller. The nutrient medium flow rate can then be adjusted based on the biomass concentration transmitted by the biomass sensor. The controller can be configured to adjust the medium flow rate based on the information received from the biomass sensor.

[0009] The biomass sensor can include, for example, a capacitance sensor adapted to be mounted within the perfusion bioreactor. Alternatively, the biomass sensor may include an optical cell counter. In one embodiment, for example, the perfusion bioreactor can be in fluid communication with an automatic sampling system. The automatic sampling system can continuously or periodically remove samples from the perfusion bioreactor for testing using the biomass sensor. In one embodiment, the biomass sensor can take biomass concentration readings at least every 6 hours, e.g., at least every 4 hours, e.g., at least every 30 minutes, e.g., at least every 10 minutes. The biomass concentration measurements can be provided to a controller, which can include an algorithm for determining the nutrient medium flow rate. For example, the nutrient medium flow rate can be determined according to the following relationship: P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); P can be adjusted based on the perfusion rate (expressed in mL fed / mL bioreactor / day). Generally, the controller can be configured to increase the nutrient medium flow rate to the perfusion bioreactor as the cell density in the bioreactor increases.

[0010] In addition to controlling the nutrient medium flow rate supplied to the perfusion bioreactor, the fluid medium flow rate removed from the perfusion bioreactor can also be controlled. For example, in one embodiment, the amount of biomass and fluid medium in the perfusion bioreactor can be determined. Based on this amount, the rate at which fluid medium is removed from the perfusion bioreactor can be selectively increased or decreased. In one embodiment, for example, the amount of biomass and fluid medium in the perfusion bioreactor can be determined using a weighing device. The weighing device can be in communication with a controller. Based on weight information from the weighing device, the controller can be configured to control a pump device in fluid communication with the perfusion bioreactor to selectively increase or decrease the rate at which fluid medium is removed. For example, the rate at which fluid medium is removed can be performed in such a way that the volume within the perfusion bioreactor, including the fluid medium and cell culture, remains constant during the process.

[0011] Instead of a metering device, the process and system may use a volume level indicator to determine the amount of fluid medium in the perfusion bioreactor. The volume indicator may also be placed in communication with a controller to automatically control the rate of fluid medium removal from the perfusion bioreactor.

[0012] The perfusion bioreactor generally can have a volume of about 10 liters to about 4000 liters, or about 1000 liters to about 4000 liters. During the inoculation process, the cell culture is grown in the perfusion bioreactor at a density of about 10 x 10 6 More than 30 x 10 cells / mL, e.g., approximately 30 x 10 6More than 50 x 10 cells / mL, e.g., approximately 50 x 10 6 More than 70 x 10 cells / mL, e.g., approximately 70 x 10 6 In one embodiment, cell cultures can reach cell densities of over 100 x 10 cells / mL. 6 Cell densities of over 1000 x 10 cells / mL can be achieved. 6 The cell culture can remain in the perfusion bioreactor for about 3 days to about 12 days.

[0013] The process of the present disclosure can further include 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 can be a batch-fed reactor and can have a volume of about 10 L to about 30,000 L. The second bioreactor can have a volume 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 can be about 1:3 to about 1:40, such as about 1:4 to about 1:10. The cell culture can remain in the second bioreactor for a period of less than about 12 days, e.g., less than about 11 days, e.g., less than about 10 days, and still be able to produce a desired amount of bioproduct.

[0014] The present disclosure also relates to a system for producing an inoculum for a subsequent cell culture production process. Accordingly, in a second aspect, the present invention also relates to a system for producing an inoculum for a subsequent cell culture production process, the system comprising: a perfusion bioreactor; a nutrient medium supply in fluid communication with the perfusion bioreactor for supplying nutrient medium to the perfusion bioreactor for growing a cell culture; an outlet for removing the fluid medium from the perfusion bioreactor; a pump device in fluid communication with the outlet of the perfusion bioreactor for removing 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 capacitance sensor, in fluid communication with the perfusion bioreactor for determining a biomass concentration within the perfusion bioreactor; a controller in communication with the biomass sensor and the metering device, the controller being configured to control the nutrient medium supply to increase or decrease a flow rate of nutrient medium supplied to the perfusion bioreactor based on information received from the biomass sensor, and to control the pump device to increase or decrease a flow rate of fluid medium removed from the perfusion bioreactor based on information received from the metering device; The controller is for the system to control the weight flow rate of nutrient medium to the perfusion bioreactor based on the following relationship: P=K*φ as described above.

[0015] The system includes a nutrient medium supply in fluid communication with the perfusion bioreactor. The nutrient medium supply is for supplying a nutrient medium to the perfusion bioreactor for growing a cell culture. The perfusion bioreactor can also include an outlet for removing a fluid medium from the perfusion bioreactor. A pump device can be in fluid communication with the outlet for controlling the flow of the fluid medium from the perfusion bioreactor. The system can further include a weighing device for monitoring a weight of the perfusion bioreactor and a biomass sensor in communication with the perfusion bioreactor for determining a biomass concentration in the perfusion bioreactor. A controller can be in communication with the biomass sensor and the weighing device. The controller can be configured to control the nutrient medium supply to increase or decrease a flow rate of the nutrient medium supplied to the perfusion bioreactor based on information received from the biomass sensor. The controller can also be configured to control the pump device to increase or decrease the flow rate of the fluid medium from the perfusion bioreactor based on information received from the weighing device. The controller may comprise, for example, one or more microprocessors.

[0016] As noted above, the processes and systems of the present disclosure are particularly well suited for producing inoculum for subsequent cell culture production processes.

[0017] Thus, in a third aspect, the present invention provides a cell culture production process comprising: Producing an inoculum by the method of the first aspect of the invention comprising a host cell expressing a bioproduct; introducing an inoculum into a production bioreactor and a cell culture into a perfusion bioreactor; Culturing the host cell to produce the bioproduct; harvesting the bioproduct from the cell culture; Optionally, subjecting the bioproduct to one or more purification steps.

[0018] Alternatively, however, the processes and systems of the present disclosure can be used to produce a bioproduct without transferring to a subsequent batch bioreactor. For example, in one embodiment, a cell culture can be incubated in a perfusion bioreactor to reach a desired cell density. The cell culture can then be fed to a purification process and / or a process for harvesting a bioproduct from the cell culture.

[0019] Other features and aspects of the disclosure are discussed in more detail below.

[0020] A full and enabling disclosure of the present disclosure is set forth more particularly in the remainder of the specification, including reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 illustrates one embodiment of a perfusion bioreactor system according to the present disclosure for producing inoculum for downstream cell production. [Figure 2]FIG. 1 illustrates one embodiment of a system for producing inoculum and transferring the inoculum to a large-scale bioreactor for producing a bioproduct. [Figure 3] 1 is a graphical representation illustrating various benefits and advantages of the present disclosure. [Figure 4A] 10 is an exemplary graph of variable feeding, according to embodiments herein. [Figure 4B] 10 is an exemplary graph of variable feeding, according to embodiments herein. [Figure 5] 1 is a graph showing concentrated nutrient feed rate as a function of perfusion medium feed rate normalized by biovolume. [Figure 6] 12 is a graph showing concentrated nutrient feed rate as a function of perfusion medium feed rate normalized per cell. [Figure 7] 1 is a graph showing concentrated nutrient feed rate as a function of perfusion medium feed rate normalized by biovolume. [Figure 8] 12 is a graph showing concentrated nutrient feed rate as a function of perfusion medium feed rate normalized per cell. [Figure 9] 1 is a graph illustrating a constant feeding strategy per biovolume, according to embodiments herein. [Figure 10] 1 is a graph showing a selection of conditions for investigating biovolume-specific perfusion rates on cell culture performance. [Figure 11A] 1 is a graph showing medium consumption and cell density for four BVSPR conditions. [Figure 11B] 1 is a graph showing medium consumption and cell density for four BVSPR conditions. [Figure 12] 1 is a graph showing the cell densities of five cell clones cultured under BVSPR conditions at 7 mL / mL / day. [Figure 13] 1 is a graph showing the scale-up of the N-1 process from bench scale to a 50 L bioreactor.

[0022] [Mode for Carrying Out the Invention] It should be understood by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present disclosure.

[0023] The present disclosure relates generally to processes and systems for producing biological products. More specifically, the present disclosure relates to processes and systems for producing an inoculum that is transferred to a large-scale bioreactor for the production of biological products. The inoculum is grown in a perfusion bioreactor, and the goal of the process is to produce a rapidly increasing biomass over the course of the culture. Process control is used to carefully control the rate at which a nutrient medium is fed into the perfusion bioreactor and the rate at which fluid medium is removed from the perfusion bioreactor. According to the present disclosure, the nutrient medium flow rate and the removal rate are periodically and / or continuously adjusted to maintain an optimal feed rate per biomass in the perfusion bioreactor while maintaining a constant volume or constant mass condition. In one embodiment, the process can be fully automated to adjust the nutrient medium feed rate based on biomass concentration measurements made in real time. For example, the nutrient medium feed rate can be adjusted so that it is directly proportional to the current biomass in the reactor.

[0024] In addition to generating inoculum, the disclosed processes and systems can also be used to produce cell cultures that do not require additional incubation time. For example, the disclosed perfusion bioreactor can produce cell cultures with cell densities where a bioproduct can be harvested directly from the perfusion bioreactor. Alternatively, the cell culture incubated in the perfusion bioreactor can be fed to a purification process for later harvesting the bioproduct.

[0025] The disclosed methods and systems can be applied to any suitable cell culture product. For example, the disclosed methods are particularly well suited for the production of biopharmaceuticals, such as biotherapeutic proteins. For example, biotherapeutic proteins are produced from genetically modified mammalian cells. In one embodiment, the cell culture can be produced via recombinant gene expression in a cellular host. Such production can be from established cell lines, such as CHO, NSO, or PER.C6, for example. These cells can express a protein of interest and subsequently secrete the protein into the medium. However, it should be understood that the disclosed processes and techniques are not limited to protein production, and any suitable cell culture can be subjected to the control described herein.

[0026] As noted above, in one embodiment, the present disclosure generally relates to systems and processes for producing inoculum that can be transferred to larger bioreactors, such as commercial-scale bioreactors. According to the present disclosure, inoculum can be prepared that contains microorganisms that are compatible and well-suited for further growth in a production bioreactor at relatively high cell densities and viable cell counts. For example, the disclosed processes and systems can achieve high levels of viable biomass in a physiological state suitable for use as an inoculum. In addition to producing proteins, the disclosed processes and systems can be used to produce antimicrobials, enzymes, beverages, drugs, toxins, vitamins, amino acids, and the like.

[0027] Referring to Figure 1, one embodiment of a perfusion bioreactor system that can be used to generate an inoculum according to the present disclosure is shown. The diagram shown in Figure 1 is for illustrative purposes only and is not intended to limit the types of perfusion bioreactor systems that can be used to generate quality attribute information. Generally, perfusion bioreactor systems can be configured as highly automated process development platforms. Perfusion bioreactor systems can be used to produce inoculum with very high cell densities and / or viable cell counts.

[0028] As shown in FIG. 1 , the perfusion bioreactor system includes a perfusion bioreactor 10. The perfusion bioreactor 10 may include any suitable bioreactor, depending on the cell culture being grown. For example, the perfusion bioreactor 10 may include a fermentor, a stirred tank reactor, a wave bioreactor, a rocking reactor, etc. The perfusion bioreactor 10 in the embodiment shown in FIG. 1 comprises a hollow vessel or container including a bioreactor volume 12 for receiving a cell culture in a fluid growth medium. The perfusion bioreactor 10 may be mounted in association with a rotatable shaft coupled to an agitator 13 for agitating the cell culture contained within the bioreactor volume 12.

[0029] The perfusion bioreactor 10 can be made from a variety of materials. For example, the bioreactor 10 can be made from a metal, such as stainless steel, and can be designed to be reused. Alternatively, the perfusion bioreactor 10 may comprise a single-use bioreactor made from a rigid polymer or flexible polymer film. The walls of the bioreactor can be freestanding, for example, if made from a rigid polymer. Alternatively, the bioreactor 10 can be made from a flexible polymer film or form-fitting material that is liquid-impermeable and can have an internal hydrophilic surface. In one embodiment, the perfusion bioreactor 10 can be made from a flexible polymer film designed to be inserted into a rigid structure, such as a metal container, to assume a desired shape.

[0030] The perfusion bioreactor 10 can have any suitable volume. For example, the volume of the perfusion bioreactor 10 can generally be greater than about 1 L, e.g., greater than about 5 L, e.g., greater than about 10 L. In embodiments, the volume of the perfusion bioreactor 10 is generally less than about 400 L, e.g., less than about 250 L, e.g., 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, e.g., greater than 500 L, e.g., greater than 750 L, e.g., greater than 1000 L, e.g., greater than 1500 L, and generally less than about 4000 L, e.g., less than about 3000 L, e.g., between about 10 L and about 4000 L.

[0031] The perfusion bioreactor 10 can also include various other components and equipment that enable the cultivation and growth of biological cells, such as baffles, spargers, gas supplies, heat exchangers, etc. Additionally, the perfusion bioreactor 10 can be in communication with various sensors, such as pH sensors, gas sensors, temperature sensors, etc.

[0032] The perfusion bioreactor 10 is designed to continuously receive various inputs, such as nutrient medium, and to continuously remove spent medium to maintain a pseudo-steady-state condition within the cell culture contained within the bioreactor 10. For example, in one embodiment, the perfusion bioreactor 10 operates to maintain a relatively constant volume of cell culture and medium. For example, the perfusion bioreactor 10 can be operated such that the volume within the bioreactor does not change by more than 10%, e.g., by about 8% or less, e.g., by about 5% or less, e.g., by about 3% or less.

[0033] There are a variety of different methods for removing spent medium from the perfusion bioreactor 10 without depleting the biological cells. For example, in one embodiment, the perfusion bioreactor can include an attachment device, such as a capillary fiber or membrane, that prevents cells from escaping by binding them. In other embodiments, the perfusion bioreactor 10 can include a filter device 15 that maintains a desired cell density in the bioreactor. By continuously removing spent medium from the perfusion bioreactor 10 and replacing it with fresh medium, nutrient levels can be controlled and maintained to vary growth conditions within the bioreactor. Additionally, cellular waste products can be removed in a controlled manner to avoid toxicity.

[0034] The perfusion bioreactor 10 can include multiple ports. The ports can allow for supply and supply lines to and from the bioreactor 10 for adding and removing fluids and other materials. Additionally, one or more ports may be connected to one or more probes for monitoring conditions within the perfusion bioreactor 10.

[0035] In the embodiment shown in FIG. 1 , for example, perfusion bioreactor 10 includes outlet port 14 and inlet port 18. Outlet port 14 is for continuous or periodic removal of fluid medium from perfusion bioreactor 10. Outlet port 14 can be in fluid communication with pump 22 for controlling the flow rate. Meanwhile, inlet port 18 can be in fluid communication with nutrient medium supply 16 and pump 20. Pump 20 can be designed to pump a controlled amount of nutrient medium supply into perfusion bioreactor 10 via inlet port 18. In one embodiment, only a single inlet port 18 is needed to supply nutrient medium to perfusion bioreactor 10. However, in other embodiments, multiple ports may be used to supply nutrient medium, basal medium, and / or other optional components, such as pH adjusters, gases such as oxygen, nitrogen, and carbon dioxide.

[0036] As used herein, nutrient medium or nutrients refers to any fluid, compound, molecule, or substance capable of increasing the mass of a bioproduct, such as one that an organism can use to live, grow, or otherwise add biomass. For example, nutrient supplies can include gases such as oxygen or carbon dioxide used for respiration or any type of metabolism. Other nutrient media can include a carbohydrate source. Carbohydrate sources include complex and simple sugars such as glucose, maltose, fructose, galactose, and mixtures thereof. Nutrient media can also include amino acids. Amino acids can 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.

[0037] The nutrient medium may also contain one or more vitamins. Vitamins that may be included in the nutrient medium include group 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 supply proteins and peptides to the bioreactor. Proteins and peptides include, for example, albumin, transferrin, fibronectin, fetuin, and mixtures thereof. Growth media within the present disclosure may also include growth factors and growth inhibitors, trace elements, inorganic salts, hydrolysates, and mixtures thereof. Trace elements that may be included in the growth medium include trace metals. Examples of trace metals include cobalt, nickel, and the like.

[0038] As shown in FIG. 1, the system further includes a biomass sensor 24. The biomass sensor 24 can be used to assay the biomass concentration within the perfusion bioreactor 10. As used herein, "biomass concentration" refers to the volume of cells (typically viable cells) contained within the bioreactor relative to the total volume (mL / mL) of the bioreactor 10, which refers to the total filled volume (liquid volume) of the bioreactor, i.e., containing the fluid medium, cells, cell debris, etc. For example, the biomass sensor 24 can be a capacitance probe. Cells contained within the perfusion bioreactor 10 with intact plasma membranes function as capacitors under the influence of an electric field. The non-conductivity of the plasma membrane allows for the accumulation of charge. 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 resulting measurements can be used to derive the biomass content (e.g., viable cell volume) present in the perfusion bioreactor 10, as well as other cellular characteristics such as cell diameter (see, e.g., U.S. Pat. No. 9,568,449, the disclosure of which is incorporated herein by reference in its entirety, including disclosure of biomass content measurements). The biomass sensor 24 can be a repetitively used device or a single-use device. In one embodiment, for example, the biosensor can be a patch sensor that can be used once and then disposed of.

[0039] When using a capacitance probe, the capacitance probe may operate in a frequency range of about 500 KHz to about 20,000 KHz. The capacitance measurement range is about 0 to about 400 pF / cm. The conductivity may range from about 1 to about 40 mS / cm. Suitable methods for measuring biomass concentration using a capacitance probe are provided in the Examples. Suitable capacitance probes include the BioPAT® ViaMass from Sartorius Stedim Biotech (e.g., for single-use applications) and the Futura 12 mm Probe from Aber Instruments Ltd. (e.g., for multi-use applications).

[0040] In addition to a capacitance probe, the biomass mass sensor 24 can be any other suitable device capable of monitoring or determining biomass concentration or cell number. For example, in an alternative embodiment, the biomass sensor 24 can be an optical cell counter. For example, optical cell counters are commercially available from ThermoFisher Scientific under the tradenames COUNTESS II or COUNTESS II FL Automated Cell Counter. Optical cell counters include autofocus and counting algorithms that identify cells within a population to determine biomass concentration. Methods for determining biomass concentration (biovium) using optical cell counters are known in the art and utilize measurements of cell number combined with cell diameter from an optical device to calculate biomass concentration. Additional methods for determining biomass concentration include, for example, microscopy-based methods.

[0041] As shown in Figure 1, a biomass sensor 24 can be included within volume 12 of perfusion bioreactor 10 for intimate contact with the cell culture contained within the bioreactor. In the embodiment shown in Figure 1, for example, biomass sensor 24 can be a capacitance probe as described above.

[0042] Alternatively, the system can include at least one sample collection subsystem that obtains biomass samples from the perfusion bioreactor and analyzes the samples for biomass concentration and / or other constituents. For example, in one embodiment, the perfusion bioreactor 10 can be in fluid communication with an automated sampling and testing system. The biomass sample can be fed to a sterile autosampler, which can then route the sample to a liquid-handling robot that automates sample preparation, if necessary. The sampling and testing system can measure and monitor any parameter within the cell culture, such as cell count 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.

[0043] When biomass concentration measurements are performed using an automated sampling system, the biomass sensor can include a capacitance probe or an optical cell counter.

[0044] The biomass sensor 24 may take a reading 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 may continuously monitor the biomass concentration within the perfusion bioreactor 10.

[0045] The biomass sensor 24 can be in communication with the controller 26 and the pump 20. The controller 26 may include one or more programmable devices, such as, for example, 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 nutrient medium flow rate to the perfusion bioreactor 10 by controlling the pump 20.

[0046] In conventional perfusion bioreactors, the perfusion rate generally remains within a relatively narrow range. However, in the process of the present disclosure, the perfusion rate, or the rate at which nutrient medium is fed into the perfusion bioreactor 10, is constantly varied as the biomass or cell culture within the perfusion bioreactor 10 rapidly increases in order to produce an inoculum of dramatically improved cell density and viable cell count.

[0047] For example, according to the present disclosure, the flow rate of nutrient medium from nutrient medium supply 16 is varied in a manner that is directionally proportional to the current amount of biomass contained within perfusion bioreactor 10, as determined by biomass sensor 24. Biomass sensor 24 can obtain real-time biomass concentration measurements, which are provided to controller 26, allowing for full automation of nutrient medium flow rate through the bioreactor. For example, during the process, the nutrient medium flow rate increases in response to the viable biomass concentration. For example, in one embodiment, controller 26 can be programmed with an algorithm that determines the nutrient flow rate based on information received from biomass sensor 24. In one embodiment, the algorithm includes the following: P=K*φ / V where K is the biovolume-specific feed rate and has units of mL / % biovol / day; Biovolume (denoted as φ) can be based on the fraction of the reactor volume (fill volume) that is inside the cell membrane of the cells, V is the vessel volume in mL, and P is the total bioreactor perfusion feed rate in vessel volumes / day. This relationship is P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane (fill volume), expressed as a percentage or ratio (mL biovolume / mL bioreactor); It can also be expressed as (P is the perfusion rate expressed in mL feed / mL bioreactor / day). Another way to express this, especially when using capacitance to derive biomass, is as a percentage based on dividing the intracellular volume of viable cells (VCV) by the total filled volume of the bioreactor (i.e., occupied by culture medium and cells / debris, etc.).

[0048] The above relationship can be determined for a particular cell culture growing in perfusion bioreactor 10 based on a variety of other process conditions. K in the above equation can be determined experimentally or by theoretical calculation. For example, in one embodiment, K is approximately 1×10 -9 ~Approx. 250×10 -9 , appropriately, about 1 x 10 -9 ~Approx. 50×10 -9 , or approximately 1 × 10 -9 ~Approx. 20×10 -9 , for example, about 1 x 10 -9 ~About 10×10 -9 , about 4×10 -9 ~Approx. 9×10 -9 , or approximately 7 × 10 -9The amount of nutrient used can vary. As described herein, it has surprisingly been found that the use of biovolume (the percentage of reactor volume inside the cell membrane) provides a better predictor of required nutrient medium flow compared to measurements made using viable cell density (VCD) (i.e., cell number). VCD assumes a constant nutrient consumption rate per cell, so it is simply the number of cells that matters. However, because larger cells consume more nutrients than smaller cells, a more accurate predictor of nutrient use, as described herein, is based on biovolume.

[0049] In addition to controlling the nutrient medium supply rate to the perfusion bioreactor 10, the disclosed process and system shown in FIG. 1 may also be configured to control the rate at which fluid medium is removed from the bioreactor 10 through the outlet 14 using a pump 22. For example, in one embodiment, the system may include a weighing device 28, such as a load cell. The weighing device 28 may monitor the weight of the fluid medium and biomass contained within the perfusion bioreactor 10. As shown in FIG. 1, the weighing device 28 may be in communication with the controller 26 and the pump 22. The controller 26 may be configured to control the amount of fluid medium removed from the perfusion bioreactor 10 using the pump 22 based on information received from the weighing device 28. In addition to relying on information received from the weighing device 28, the controller 26 may also take into account increases in the nutrient medium flow rate to the bioreactor when determining the rate at which fluid medium is removed from the bioreactor.

[0050] In addition to metering device 28, the system may also include other devices for determining the amount of fluid medium within perfusion bioreactor 10. For example, in an alternative embodiment, the system may include a volume level indicator that monitors the volume of fluid medium within bioreactor 10. The volume level indicator may also be mounted in communication with controller 26.

[0051] In one embodiment, the controller 26 can be configured to operate the perfusion bioreactor 10 to maintain a relatively constant volume. For example, the volume can change by about 20% or less, e.g., about 15% or less, e.g., about 10% or less, e.g., about 5% or less, e.g., about 2% or less, during the process.

[0052] Through the process and system of the present disclosure, inocula for downstream cell production can be produced at very high cell densities and viable cell numbers. For example, a cell culture in a perfusion bioreactor can increase in cell density or biomass concentration by more than about 30% per day, for example, more than about 40% per day, for example, more than about 50% per day, for example, more than about 60% per day, for example, more than about 70% per day, for example, more than about 80% per day, for example, more than about 90% per day, for example, more than about 100% per day, for example, more than about 110% per day, for example, more than about 120% per day. For example, biomass concentration can increase by more than 150% per day, for example, more than 200% per day, for example, more than 250% per day.

[0053] After a desired amount of growth has occurred in the perfusion bioreactor, the inoculum or cell culture is transferred to a larger downstream bioreactor for continued growth and harvest of the bioproduct. The cell culture can remain in the perfusion bioreactor for a sufficient time to achieve a desired cell density or biomass concentration. For example, according to the present disclosure, the cell culture or inoculum can be approximately 10 x 10 6 More than 30 x 10 cells / mL, e.g., approximately 30 x 10 6 More than 50 x 10 cells / mL, e.g., approximately 50 x 10 6 cells / mL, e.g., even 70 x 10 6 In embodiments, the cell density achieved in the perfusion bioreactor can be greater than 100×10 cells / mL. 6 cells / mL, 200×10 6 cells / mL, 220×10 6 cells / mL, 250×106 cells / mL or more, however, the desired cell density may depend on various process conditions and the type of cell culture being produced.

[0054] Generally, any suitable inoculum can be produced using the processes and systems of the present disclosure. In one embodiment, for example, the inoculum may comprise mammalian cells.

[0055] Increasing the cell density and / or viable cell count of the inoculum can yield many benefits and advantages during downstream production. For example, a higher cell density inoculum can reduce the residence time of the cell culture in a larger downstream production bioreactor. Reducing the incubation time in the production bioreactor directly impacts process efficiency. For example, large, commercial-scale bioreactors occupy a large portion of the floor space of a production facility. Thus, the incubation time in the production bioreactor is the limiting event in production efficiency. However, through the processes and systems disclosed herein, incubation time can be dramatically reduced in the downstream production bioreactor, resulting in increased space-time yield. In fact, these benefits and advantages are achieved even with longer incubation times for the inoculum in the perfusion bioreactor.

[0056] Referring to FIG. 2, one embodiment of a production process for producing a bioproduct is shown. As shown, in one embodiment, an inoculum 40 can first be provided in a small cell culture vessel 50. For example, the cell culture vessel 50 can be a shaker flask expansion device. The inoculum can be grown to a limited extent in these dedicated incubator vessels. For example, the cell culture vessel 50 can generally have a volume greater than about 0.5 L, e.g., greater than about 1 L, e.g., greater than about 2 L, and generally less than about 5 L, e.g., less than about 4 L, less than about 3 L.

[0057] From the cell culture vessel 50, the inoculum is then fed into the perfusion bioreactor 10 of the present disclosure. As shown in FIG. 2, the perfusion bioreactor 10 can be a rocking bioreactor, a wave bioreactor, or a stirred tank bioreactor. In one embodiment, the perfusion bioreactor 10 can have a volume of about 5 L to about 4000 L, e.g., about 10 L to about 3000 L, e.g., about 50 L to about 2000 L. The perfusion bioreactor 10 can be a stainless steel vessel or can be a disposable bag-type bioreactor used as a lining within a holding vessel.

[0058] The incubation time in the perfusion bioreactor 10 can vary depending on the inoculum being produced and the desired final cell density. For example, the incubation time of the inoculum with the perfusion bioreactor 10 can generally be greater than about 3 days, e.g., greater than about 5 days, e.g., greater than about 7 days, e.g., greater than about 9 days, and generally less than about 15 days, e.g., less than about 12 days, e.g., less than about 11 days. As noted above, the perfusion bioreactor 10 is particularly well-suited for producing inoculum of dramatically improved cell density and viable cell number.

[0059] From the perfusion bioreactor 10, the inoculum is then fed to a production bioreactor 60 for producing a bioproduct. In one embodiment, for example, the production bioreactor 60 can have a volume greater than about 500 L, e.g., greater than about 600 L, e.g., greater than about 700 L, and generally less than about 30,000 L, e.g., less than about 20,000 L, e.g., less than about 10,000 L. Generally, 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 can be about 1:3 to 1:40, e.g., about 1:4 to about 1:10 (e.g., a 4,000 L perfusion bioreactor to a 20,000 L production (e.g., batch-fed) bioreactor).

[0060] Because the cell density of the inoculum fed to the production bioreactor 60 is increased, the incubation time in the bioreactor 60 can be significantly reduced. For example, conventional systems typically require an incubation time of 15 days or more. However, the incubation time in the production bioreactor 60 according to the present disclosure can be less than about 13 days, e.g., less than about 12 days, e.g., less than about 11 days, e.g., less than about 10 days, e.g., less than about 9 days, e.g., less than about 8 days. The incubation time is generally greater than about 3 days, e.g., greater than about 5 days. Reducing the incubation time in the production bioreactor 60 has a significant impact on improving the efficiency of the overall process.

[0061] By producing a higher cell density inoculum, higher titers can be produced in the production bioreactor in a shorter period of time by eliminating non-productive start-up days in the production bioreactor. To illustrate this effect, Figure 3 is a graphical representation of some of the benefits of the process of the present disclosure. The graph in Figure 3 shows viable cell density and titer over time in the production bioreactor. Sample No. 1 was obtained when the cell density of the inoculum fed to the production bioreactor was 5 x 10 5 However, sample No. 2, produced in accordance with the present disclosure, was 10×10 cells / mL. 6 The figures represent feeding an inoculum to the production bioreactor at a cell density of 1000 cells / mL. As shown, Sample No. 2 produced dramatically superior growth rates and an overall higher titer. For example, after 15 days, Sample No. 1 reached a titer of 4.95 g / L, while Sample No. 2 reached a titer of 6.13 g / L in 12 days. Dividing the titer by the incubation time yields the space-time yield. Sample No. 1 produced a space-time yield of 0.3 g / L / day, while Sample No. 2 produced a space-time yield of 0.51 g / L / day. Thus, the process according to the present disclosure produced a 50% increase in space-time yield.

[0062] As noted above, the perfusion bioreactor of the present disclosure can produce significant and dramatic increases in cell density compared to many conventional processes. Indeed, through experimental procedures, the perfusion bioreactor of the present disclosure was able to produce cell densities of up to 80×10 6 cells / mL, e.g., 90 x 10 6 In fact, the perfusion bioreactor of the present disclosure can produce cell cultures with cell densities of 100×10 cells / mL. 6 It has demonstrated the ability to generate cell densities of >100 cells / mL.

[0063] In addition to producing an inoculum through increased cell density, the perfusion bioreactor of the present disclosure can also be used to produce an end product. For example, in one embodiment, the perfusion bioreactor of the present disclosure can be used to incubate a cell culture and harvest a bioproduct directly from the cell culture. In one embodiment, the cell culture produced in the perfusion bioreactor can then be fed to a downstream purification process to harvest the bioproduct. Thus, the present invention also provides a cell culture production process, comprising: Producing an inoculum by the method of the first aspect of the invention comprising a host cell expressing a bioproduct; introducing an inoculum into a production bioreactor and a cell culture into a perfusion bioreactor; Culturing the host cell to produce the bioproduct; harvesting the bioproduct from the cell culture; Optionally, subjecting the bioproduct to one or more purification steps.

[0064] In one embodiment, the inoculum is at least 5 x 10 6 cells / mL, e.g., at least 8 or 10 x 10 6 The culture medium is introduced into the production bioreactor to a final density of 1000 cells / mL, which can represent, for example, a 4- to 10-fold dilution of the inoculum from the N-1 process of the present invention.

[0065] In embodiments, the cells express or produce a product, such as a recombinant therapeutic or diagnostic product. Examples of products produced by the cells include, but are not limited to, antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to antibodies, such as, for example, 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., anti-cancer oncolytic viruses, viral vectors for gene therapy and viral immunotherapy), cellular 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, etc.) or DNA (e.g., plasmid DNA, etc.), antibiotics, or amino acids. In embodiments, the devices, facilities, and methods can be used in the manufacture of biosimilars.

[0066] As noted above, in embodiments, the devices, facilities, and methods enable the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells (e.g., yeast cells or filamentous fungal cells, etc.), or prokaryotic cells, such as Gram-positive or Gram-negative cells, and / or eukaryotic cells or eukaryotic cellular products, e.g., proteins, peptides, antibiotics, amino acids, nucleic acids (e.g., DNA or RNA), synthesized by eukaryotic cells in a large-scale manner. Unless otherwise specified herein, the devices, facilities, and methods can include any desired capacity or production capacity, including, but not limited to, bench-scale, pilot-scale, and full-production-scale capabilities.

[0067] Furthermore, unless otherwise specified herein, the devices, facilities, and methods can include any suitable reactor(s), including, but not limited to, stirred tank, air-lift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, a "reactor" can include a fermentor or fermentation unit, or any other reaction vessel, and the term "reactor" is used synonymously with "fermentor." For example, in some embodiments, an exemplary bioreactor unit can perform one or more or all of the following: feeding nutrients and / or carbon sources, injecting a suitable gas (e.g., oxygen), inflow and outflow of fermentation or cell culture medium, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, agitation (e.g., stirring), and / or cleaning / sterilization. Exemplary reactor units, such as fermentation units, can include multiple reactors within the unit; for example, a unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors within each unit, and / or a facility can include multiple units with single or multiple reactors within the facility. In various embodiments, the bioreactors can be suitable for batch, semi-fed-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactors can have a volume of from about 100 mL to about 50,000 L.Non-limiting examples include 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, Included are volumes of 650 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 multi-use, single-use, disposable, or non-disposable, and can be formed from any suitable material, including stainless steel (e.g., 316L or any other suitable stainless steel) and metal alloys such as Inconel, plastic, and / or glass.

[0068] Once product biosynthesis by the production cells has progressed to a satisfactory point, the product can be harvested, for example, by removing the medium and separating the supernatant from the cells and cell debris. The product can be subjected to one or more purification / processing steps to obtain a purified product, such as affinity chromatography, ion exchange chromatography, filtration, and / or viral inactivation. The product can also be combined with one or more pharmaceutically acceptable carriers, excipients, or diluents, for example, with one or more of a buffer, surfactant, stabilizer (such as trehalose, sucrose, glycerol), amino acid (such as glycine, histidine, arginine), metal ion / chelating agent, salt, and / or preservative, to produce a composition, such as a formulated pharmaceutical composition.

[0069] The devices, facilities, and methods described herein are suitable for culturing any desired cell line, including prokaryotic and / or eukaryotic cell lines. Furthermore, in embodiments, the devices, facilities, and methods are suitable for culturing suspension or anchorage-dependent (adherent) cells, and are suitable for manufacturing operations configured for the production of pharmaceutical and biopharmaceutical products, such as polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and / or viruses, such as those used in cell and / or virus therapy. In one embodiment, the host cell is a mammalian cell. Examples of species from which the host cell can be derived include human, mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, and cat. In an embodiment, the host cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the host cell is a CHO-K1 cell, a CHOK1SV® cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHO-S, a CHO GS knockout cell (a CHO cell in which all endogenous copies of the glutathione synthetase (GS) gene have been inactivated), a CHOK1SV® FUT8 knockout cell, a CHOZN, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GS-KO cell) is, for example, a CHOK1SV® GS knockout cell (GS Xceed®-CHOK1SV GS-KO® from Lonza Biologics, Inc.). A CHO FUT8 knockout cell is, for example, a Potelligent® CHOK1SV® FUT8 knockout (Lonza Biologics, Inc.).

[0070] In embodiments, unless otherwise specified herein, the devices, facilities, and methods described herein can also include any suitable unit operations and / or equipment not specifically mentioned, e.g., operations and / or equipment for the separation, purification, and isolation of such products. Any suitable facilities and environments can be used, such as traditional stick-built facilities, modular, mobile, and temporary facilities, or any other suitable construction, facility, and / or layout. For example, in some embodiments, modular cleanrooms can be used. Furthermore, unless otherwise specified, the devices, systems, and methods described herein can be housed and / or performed in a single location or facility, or can be housed and / or performed in separate or multiple locations and / or facilities.

[0071] Example Example 1: Biovolume-based perfusion reactor feeding predicts good culture performance and is superior to cell number-based feeding. Capacitive measurement of biomass concentration (biomass volume) Culture biomass concentration (biomass volume) 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. Capacitance values ​​were correlated with biovolume (biomass concentration) in calibration experiments, in which daily samples were extracted from growing bioreactor cultures at different viable cell concentrations. Viable cell concentration and mean cell diameter were determined for each sample using a Nova Bioprofile Flex (Nova Biomedical, Waltham, MA). Viable biovolume was determined from the aforementioned measurements from the Flex by assuming a spherical cell shape as follows:

[0072]

number

[0073]

number

[0074] Special note about biovolume: The biovolume fraction φ, in mL / mL, is analogous to concentration, VCC (cells / mL). Viable biovolume (mL) is analogous to total biovolume and total cell number (cells). That is, biovolume fraction and VCC are both concentrations, and viable biovolume and total cell number are sums of the total reactor volume.

[0075] Operation of perfusion cultures for supply operation space experiments Perfusion culture was performed using a single CHO cell line expressing a monoclonal antibody. 6Cells were inoculated at a viable cell concentration of 1000 cells / mL into a chemically defined basal medium (basal medium + 1.9 vol% SF102 (concentrated nutrient feed)) at pH 6.9. The dissolved oxygen concentration was maintained above 40% air saturation. Cells were allowed to expand until day 6 with supplemental feeding, which began with point perfusion at 1 vessel volume (vv) / day (perfusion medium - basal medium + 4.21 vol% SF102). As cells continued to expand, the perfusion rate was increased daily up to a maximum of 2 vv / day. Once the culture reached the desired cell concentration (or biovolume fraction, depending on the control strategy for that particular run), a capacitance-controlled cell bleed was initiated to maintain the culture at a constant cell concentration or biovolume fraction. The perfusion feed rate was manually adjusted to achieve the desired cell-specific or biovolume-specific perfusion rate, again depending on the experiment.

[0076] Manipulation of perfused N-1 cultures Perfused N-1 cultures were cultured at 0.5 x 10 6 Cells were inoculated into chemically defined basal medium at a viable cell concentration of 1000 cells / mL. Cells were allowed to expand until day 4, when perfusion was initiated. The perfusion feed rate was determined by the fraction of biovolume, as predicted by capacitance readings, as follows: P=K*φ where P is the perfusion feed rate in vv / day, K is the biovolume-specific perfusion rate (mL feed / mL biovolume / day), and φ is the biovolume fraction (mL biovolume / mL bioreactor).

[0077] To divide the cells, increase the perfusion rate accordingly to 100 x 10 6 The variable feeding was controlled down to cells / mL. An illustrative example of this variable feeding is shown in Figures 4A-B for three CHO cell clones (C1-C3).

[0078] Creating supply operation space For perfusion medium feeding, two medium feeding considerations were considered: supplementing with enough nutrients to meet the cell requirements without overfeeding, and removing waste products from the culture by dilution. The perfusion medium was separated into two components: a basal medium and a concentrated nutrient supplement. Perfusion medium with different nutrient richness was obtained by varying the amount of nutrient supplement (SF102) added to the basal medium. More nutrient supplementation resulted in a richer perfusion medium.

[0079] Perfusion media were prepared by adding various amounts of nutrient supplements. Cell-specific or biovolume-specific perfusion rates were then varied for each medium composition, and the steady-state behavior of each culture was observed. Cultures that resulted in a stable steady state for at least 5 days were considered acceptable carriers, while cultures that resulted in reduced viability or apoptosis (and usually culture crash) were considered unacceptable carriers.

[0080] For each perfusion culture condition, the cell- or biovolume-specific perfusion rate was plotted against the cell- or biovolume-specific concentrated nutrient feed rate. The biovolume- or cell-specific concentrated nutrient feed replenishment rate was determined as the biovolume- or cell-specific perfusion rate multiplied by the amount of concentrated nutrient feed (expressed as a volume fraction) added to the basal perfusion medium for that particular medium.

[0081] Implementation of feeding operation space items in perfused N-1 cultures Once we established an operational space that identified the range of perfusion rates and concentrated nutrient feed supplementation rates that resulted in acceptable (and unacceptable) culture performance (see the upper and lower dotted lines in Figure 5), we used that operational space to identify perfusion feed conditions that resulted in acceptable culture performance in unsteady-state or N-1 growth cultures. To accomplish this, we selected conditions near the center of the predefined feed operational space (see the diamonds in Figure 5).

[0082] result A series of initial perfusion cultures performed at different nutrient supply rates supported the assumption of constant nutrient consumption per cell, where unstable cultures (see X labels) were obtained at the edges of the acceptable culture performance range, with SF102 nutrient supply / cell rates either too high or too low (Figure 6).

[0083] Based on initial observations of the upper and lower limits of the proposed nutrient supply rate per cell and the assumption that these limits were constant, we attempted more intensive culture conditions that used less medium (lower CSPR) but were still predicted to operate within the optimal window of nutrient supply per cell (+ symbol, Figure 6). To our surprise, an unstable culture was instead observed under the new conditions (+), along with signs of nutrient oversupply, including changes in cell size.

[0084] The assumption of constant nutrient requirements per cell was determined to be inaccurate; instead, the required nutrients needed to be predicted per biovolume. This accounts for the fact that larger cells, due to the fact that they have more cellular machinery, require more nutrients, and conversely, smaller cells require less nutrients. When the same perfusion conditions are plotted per biovolume, the selected perfusion condition (+) is actually predicted to be on the oversupply side, correlating with what was observed (Figure 7, upper dotted line, i.e., the circled + sign, located at the upper end of the acceptable culture performance range).

[0085] Based on an updated interpretation of the supply constraints relative to the constant per biovolume assumption, we selected new conditions that fell within the predicted ideal supply range (triangle in Figure 7) of the biovolume-specific supply space. These conditions resulted in well-performing perfusion cultures.

[0086] However, when viewed as a function of CSPR, this successful condition would have been predicted to be undersupplied using the assumption of constant cell-specific nutrient supply rates (circled triangle in Figure 8, below the lower dotted line, i.e., outside the "acceptable culture performance range").

[0087] Based on this unexpected finding, a constant feeding strategy was implemented per biovolume (see Figure 9). As shown, when cells are grown in a "nutrient-rich environment," they do not exhibit acceptable growth characteristics (top image). However, cells grown in the "acceptable culture performance range" exhibit the desired cell characteristics of appropriate cell shape and density.

[0088] Implementing feed operation space in perfused N-1 process optimization Various biovolume-specific perfusion feed rates were evaluated for their impact on culture performance. Perfused N-1 cultures were completed at four biovolume-specific perfusion rates: 5, 6.2, 7, and 8.4 mL / mL / day. These conditions were selected such that intermediate conditions were predicted to provide good culture performance and adjacent conditions were near the edge of, but still within, the acceptable operating space for a single perfusion medium composition (see Figure 10).

[0089] Each culture successfully reached a cell density of at least 75 x 10^6 cells / mL, as predicted by the operating space. However, conditions near the edges of the space grew slower and consumed correspondingly more medium (8.4 and 5 BVSPR) than those in the center of the space (and 6.2 BVSPR) (see Figure 11A). Figure 11B shows the cell density (VCC) for each selected condition for CHO cell clone L1.

[0090] Finally, the optimal conditions identified in Figure 11B for the single-cell clone (7 mL / mL / day) (C1) were applied to the other four clones (C2-L5), all of which exhibited favorable growth characteristics. This supports the idea that the biovolume-specific feeding approach is a platform approach and is not limited to just one cell line (Figure 12).

[0091] In summary, we found that, surprisingly, the use of a biovolume-based model, i.e., the percentage of reactor volume inside the cell membrane (i.e., liquid volume: fluid medium, cells, cell debris, etc.), allowed us to more accurately predict the feeding conditions necessary to provide the desired perfusion culture.

[0092] Process Scaling Up Figure 13 shows that the N-1 process was successfully scaled up from benchtop scale to a 50 L single-use stirred tank reactor (pilot scale). 6 The defined target VCC of cells / mL was achieved in approximately the same culture period (approximately 9 days). In fact, this target requirement was exceeded, reaching a VCC of 70 million cells / mL on day 11. These results demonstrate that the process, including automation, could be translated to different scales and different reactor formats.

[0093] Illustrative Embodiments Embodiment 1 is a process for producing an inoculum for a subsequent cell culture production process, comprising: introducing a cell culture into a perfusion bioreactor; supplying a nutrient medium to the perfusion bioreactor at a flow rate and removing a fluid medium from the perfusion bioreactor; determining a biomass concentration over time in the perfusion bioreactor using a biomass sensor, such as a capacitance sensor, wherein the biomass sensor is in communication with a controller; and adjusting a nutrient medium flow rate to the perfusion bioreactor based on the biomass concentration sensed by the biomass sensor, wherein the controller is configured to adjust the medium flow rate based on information received from the biomass sensor, wherein the nutrient medium flow rate satisfies the following relationship: P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); where P is the perfusion rate expressed in mL fed / mL bioreactor / day), adjusted based on the process.

[0094] Embodiment 2 includes the process defined in embodiment 1, wherein the controller is configured to increase the flow rate of the nutrient medium as the biomass concentration increases.

[0095] Embodiment 3 includes the process as defined in embodiment 1 or 2, further comprising determining the amount of fluid medium in the perfusion bioreactor and, based on the amount, selectively increasing or decreasing the rate at which the fluid medium is removed from the perfusion bioreactor.

[0096] Embodiment 4 includes the process defined in embodiment 3, wherein the amount of fluid medium in the perfusion bioreactor is determined by weighing the perfusion bioreactor using a weighing device.

[0097] Embodiment 5 includes the process defined in embodiment 4, wherein the metering device is in communication with a controller, and wherein, based on weight information from the metering device, the controller is configured to control a pump device in fluid communication with the perfusion bioreactor to selectively increase or decrease the rate at which the fluid medium is removed.

[0098] Embodiment 6 includes the process defined in embodiment 5, wherein the fluid medium removed from the perfusion bioreactor is filtered to prevent biomass from being removed from the bioreactor along with the fluid medium.

[0099] Embodiment 7 includes the process defined in embodiment 3, wherein the amount of fluid medium in the perfusion bioreactor is determined by measuring the volume.

[0100] Embodiment 8 includes the process defined in any of embodiments 1-7, wherein the cell culture has a cell density, and the cell density increases over time in the perfusion bioreactor.

[0101] Embodiment 9 includes the process as defined in embodiment 8, wherein the volume containing the fluid medium and the cell culture remains constant during the process.

[0102] Embodiment 10 includes a process as defined in any one of claims 1 to 9, wherein the cell culture comprises mammalian cells.

[0103] Embodiment 11 includes the process defined in any of embodiments 1-10, wherein the biomass sensor determines the biomass concentration in the perfusion reactor at least every 6 hours.

[0104] Embodiment 12 includes the process defined in any of embodiments 1-11, wherein the perfusion bioreactor has a volume of about 10 liters to about 4000 L.

[0105] Embodiment 13 includes the process defined in any of embodiments 1-12, wherein the process further comprises transferring the cell culture from the perfusion bioreactor to a second bioreactor after the incubation period, the second bioreactor having a volume larger than the volume of the perfusion bioreactor, and wherein the volume ratio between the perfusion bioreactor and the second bioreactor is 1:3 to 1:40, e.g., about 1:4 to about 1:10.

[0106] Embodiment 14 comprises the process defined in embodiment 13, wherein the cell culture continues to grow in the second bioreactor in a fed-batch type manner.

[0107] Embodiment 15 includes the process defined in embodiment 13 or 14, wherein the cell culture remains in the perfusion bioreactor for about 3 days to about 12 days and in the second bioreactor for less than about 12 days, e.g., less than about 10 days.

[0108] Embodiment 16 is a method for culturing cells in a perfusion bioreactor, the method comprising: culturing cells in a perfusion bioreactor at a concentration of about 10×10 6 More than 30 x 10 cells / mL, e.g., approximately 30 x 10 6 More than 50 x 10 cells / mL, e.g., approximately 50 x 10 6More than 70 x 10 cells / mL, e.g., approximately 70 x 10 6 16. The process as defined in any of embodiments 1-15, wherein a cell density of more than 100 cells / mL is reached.

[0109] Embodiment 17 is a method for culturing cells in a perfusion bioreactor at a cell density of 100×10 6 17. The process as defined in any of embodiments 1-16, wherein a cell density of 100 cells / mL or more is reached.

[0110] Embodiment 18 includes the process defined in any of embodiments 1-17, wherein the cell culture has a cell density, and the cell density increases by at least 60% per day in the perfusion bioreactor.

[0111] Embodiment 19 is a system for producing inoculum for a subsequent cell culture production process, comprising: a perfusion bioreactor; a nutrient medium supply in fluid communication with the perfusion bioreactor, the nutrient medium supply for supplying nutrient medium to the perfusion bioreactor for growing a cell culture; an outlet for removing fluid medium from the perfusion bioreactor; a pump device in fluid communication with the outlet of the perfusion bioreactor for removing a controlled amount of fluid medium from the perfusion bioreactor; a weighing device for monitoring the weight of the perfusion bioreactor; and a perfusion bioreactor pump for determining a biomass concentration in the perfusion bioreactor. a biomass sensor, such as a capacitance sensor, in fluid communication with the perfusion bioreactor; and a controller in communication with the biomass sensor and the metering device, the controller configured to control a nutrient medium supply to increase or decrease a flow rate of nutrient medium supplied to the perfusion bioreactor based on information received from the biomass sensor, and to control a pump device to increase or decrease a flow rate of fluid medium removed from the perfusion bioreactor based on information received from the metering device, wherein the controller controls the flow rate of nutrient medium into the perfusion bioreactor based on the following relationship: P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or fraction (mL biovolume / mL bioreactor); P is the perfusion rate expressed in mL fed / mL bioreactor / day), based on the system.

[0112] The system of embodiment 20 as defined in embodiment 19, wherein the controller includes one or more microprocessors.

[0113] Embodiment 21 is a system as defined in embodiment 19 or 20, wherein the perfusion bioreactor has a volume of about 10 liters to about 250 liters.

[0114] Embodiment 22 is the system defined in any of embodiments 19 to 21, further comprising a second bioreactor in fluid communication with the perfusion bioreactor, the second bioreactor configured to receive the cell culture from the perfusion bioreactor, the second bioreactor having a volume greater than the volume of the perfusion bioreactor, and the volume ratio between the perfusion bioreactor and the second bioreactor is 1:3 to 1:40, for example, about 1:4 to about 1:10.

[0115] Embodiment 23 is a cell culture production process, comprising: introducing a cell culture into a perfusion bioreactor; supplying a nutrient medium to the perfusion bioreactor at a flow rate and removing a fluid medium from the perfusion bioreactor; determining a biomass concentration over time in the perfusion bioreactor using a biomass sensor, the biomass sensor being in communication with a controller; and adjusting a nutrient medium flow rate to the perfusion bioreactor based on the biomass concentration sensed by the biomass sensor, the controller being configured to adjust the medium flow rate based on information received from the biomass sensor, wherein the nutrient medium flow rate satisfies the following relationship: P=K*φ where K is the biovolume-specific perfusion rate (mL delivered / mL biovolume / day), where φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or proportion (mL biovolume / mL bioreactor), and P is the perfusion rate, expressed as mL feed / mL bioreactor / day, adjusted based on the cell culture production process.

[0116] Embodiment 24 comprises the process as defined in embodiment 23, wherein after the incubation period, the cell culture is fed to a purification process.

[0117] Embodiment 25 comprises the process as defined in embodiment 23, wherein after an incubation period, the bioproduct is harvested from the cell culture.

[0118] Embodiment 26 is a cell culture production process, comprising producing an inoculum according to any of the methods of embodiments 1 to 18, comprising host cells expressing a bioproduct; introducing an inoculum into a production bioreactor; Culturing the host cell to produce the bioproduct; harvesting the bioproduct from the cell culture; Optionally, subjecting the bioproduct to one or more purification steps.

[0119] These and other modifications and variations to the present invention may be practiced by those skilled in the art without departing from the spirit and scope of the present invention (more particularly as set forth in the appended claims). Additionally, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those skilled in the art will appreciate that the foregoing description is by way of example only and is not intended to limit the invention as further set forth in such appended claims.

Claims

1. 1. A process for producing an inoculum for a subsequent cell culture production process, comprising: introducing the cell culture into a perfusion bioreactor; supplying a nutrient medium to the perfusion bioreactor at a flow rate and removing a fluid medium from the perfusion bioreactor; determining a biomass concentration over time in the perfusion bioreactor using a biomass sensor, the biomass sensor in communication with a controller; adjusting the nutrient medium flow rate to the perfusion bioreactor based on a biomass concentration sensed by the biomass sensor, wherein the controller is configured to adjust the medium flow rate based on information received from the biomass sensor; The nutrient medium flow rate has the following relationship: P = K * φ where K is the biovolume-specific perfusion rate (mL fed / mL biovolume / day); φ is the fraction of the biovolume, the volume of the perfusion bioreactor inside the cell membrane; Expressed as a percentage or ratio (mL biovolume / mL bioreactor), P is the perfusion rate expressed in mL feed / mL bioreactor / day),

2. The process of claim 1 , wherein the biomass sensor is a capacitance sensor.

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

4. 4. The process of any one of claims 1 to 3, further comprising determining an amount of fluid medium in the perfusion bioreactor and, based on said amount, selectively increasing or decreasing a rate at which said fluid medium is removed from the perfusion bioreactor.

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

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

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

8. 8. The process of any one of claims 1 to 7, wherein cell density in the cell culture in the perfusion bioreactor increases over time, for example by at least 60% per day.

9. 9. The process of claim 1, wherein the biomass sensor determines the biomass concentration in the perfusion reactor at least every 6 hours.

10. 10. The process of any one of claims 1 to 9, wherein the perfusion bioreactor has a volume of from about 10 L to about 4000 L.

11. 11. The process of any one of claims 1 to 10, wherein the process further comprises, after an incubation period, transferring the cell culture from the perfusion bioreactor to a second bioreactor, the second bioreactor having a volume larger than the volume of the perfusion bioreactor, and wherein the volume ratio between the perfusion bioreactor and the second bioreactor is 1:3 to 1:40, for example, about 1:4 to about 1:

10.

12. 12. The process of claim 11, wherein the cell culture continues to grow in the second bioreactor in a fed-batch type manner.

13. 13. The process of claim 11 or 12, wherein the cell culture remains in the perfusion bioreactor for about 3 to about 12 days and in the second bioreactor for less than about 12 days, such as less than about 10 days.

14. The cell culture was grown in the perfusion bioreactor at approximately 10 x 10 ６ More than about 30 x 10 cells / mL ６ More than about 50 x 10 cells / mL ６ More than about 70 x 10 cells / mL ６ 14. The process of any one of claims 1 to 13, wherein a cell density of more than 100 cells / mL is reached.

15. The cell culture was grown at 100 x 10 ６ 15. The process of any one of claims 1 to 14, wherein a cell density of at least 100 cells / mL is reached.

16. The process of any one of claims 1 to 15, wherein the cell culture comprises mammalian cells.

17. 1. A system for producing an inoculum for a subsequent cell culture production process, comprising: a perfusion bioreactor; a nutrient medium supply in fluid communication with the perfusion bioreactor for supplying nutrient medium to the perfusion bioreactor for growing a cell culture; an outlet for removing fluid medium from the perfusion bioreactor; a pump device in fluid communication with the outlet of the perfusion bioreactor for removing 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 capacitance sensor, in fluid communication with the perfusion bioreactor for determining a biomass concentration within the perfusion bioreactor; a controller in communication with the biomass sensor and the metering device, the controller being configured to control the nutrient medium supply to increase or decrease a flow rate of nutrient medium supplied to the perfusion bioreactor based on information received from the biomass sensor, and to control the pump device to increase or decrease a flow rate of fluid medium removed from the perfusion bioreactor based on information received from the metering device; The controller regulates the flow rate of the nutrient medium to the perfusion bioreactor according to the following relationship: P = K * φ where K is the biovolume-specific perfusion rate (mL fed / mL biovolume / day); φ is the fraction of biovolume, the volume of the perfusion bioreactor inside the cell membrane, expressed as a percentage or ratio (mL biovolume / mL bioreactor); P is the perfusion rate expressed in mL fed / mL bioreactor / day).

18. 1. A cell culture production process comprising: Producing an inoculum by the method of any one of claims 1 to 15, comprising host cells expressing a bioproduct; introducing the inoculum into a production bioreactor; Culturing the host cells to produce the bioproduct; harvesting the bioproduct from the cell culture; Optionally, subjecting said bioproduct to one or more purification steps.

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