WO2025221977A1 - Procédé de surveillance et de régulation de la mousse à l'intérieur d'un récipient - Google Patents

Procédé de surveillance et de régulation de la mousse à l'intérieur d'un récipient

Info

Publication number
WO2025221977A1
WO2025221977A1 PCT/US2025/025130 US2025025130W WO2025221977A1 WO 2025221977 A1 WO2025221977 A1 WO 2025221977A1 US 2025025130 W US2025025130 W US 2025025130W WO 2025221977 A1 WO2025221977 A1 WO 2025221977A1
Authority
WO
WIPO (PCT)
Prior art keywords
foam
vessel
level
liquid
phase surface
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/025130
Other languages
English (en)
Inventor
Edward Hon Man CHAN
Daniel NERIA
Carmen GUAN
Christian James Randecker
Hongbo Mike Su
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genentech Inc filed Critical Genentech Inc
Publication of WO2025221977A1 publication Critical patent/WO2025221977A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/02—Means for regulation, monitoring, measurement or control, e.g. flow regulation of foam

Definitions

  • Foams may also accumulate around a vessel wall more than remote from vessel walls, therefore, even if the bioreactor was formed of a transparent material, any visual reading taken may be a false representation of the amount of foam in the vessel. Such methods further require personnel to take and act upon such a reading.
  • Efforts have been made to automate monitoring of the foam thickness within bioreactors and take indirect or non-visual readings, such as US2021/355427A1 which discloses the use of vision systems in biomanufacturing processes. Solutions have included use of cameras located within the vessel or arranged to view the contents through a viewing window, however cameras can suffer from obstruction, the output requires manual review by personnel.
  • Capacitance sensors have also been deployed in the form of adherent patches attached to the side walls of the bioreactor vessel, e.g., a single-use bag. However, such patches only provide a reading of the foam thickness adjacent to the vessel wall in the vicinity of the patches. This provides information that may not be representative of the entire liquid surface and therefore may produce erroneous or unreliable readings.
  • the bioreactor vessel is formed of a single-use bioreactor or bag it is often necessary to dispose of the capacitance sensor following the manufacturing process which increases waste and expense.
  • Previous solutions also do not address the issue that the liquid level within the bioreactor vessel may change as a result of normal manufacturing processes, for example liquid volume may change due to the introduction of reagents (such as fresh medium or supplements). This further complicates determination of the thickness of the foam on a liquid within a bioreactor.
  • the present invention seeks to at least partially address the above identified problems.
  • a method of monitoring and controlling foam within a vessel comprising a liquid comprising: a) measuring the thickness of the foam within a vessel comprising a liquid; and b) controlling the dosage of an antifoaming additive to be added to the liquid based on the measured thickness of the foam; wherein the thickness of the foam in step a is determined based on the difference between the level of a phase surface and the level of the liquid; and wherein the level of the phase surface is determined by a signal emitted towards and reflected from the phase surface.
  • a “phase surface” is the interface between two states of matter.
  • this may be the surface between the foam and gas in the head space of the vessel, or (if no foam is present) this may be the surface between the liquid and the gas in the head space of the vessel.
  • the level of the phase surface or the phase surface level means, for example, height of the phase surface cumulative with the liquid height in a vessel.
  • the phase surface may be distinct or continuous (e.g. the transition between foam state and the gas state is clearly defined in the case of distinct, or the transition between foam state and the gas state is indistinct with a transition region between the two states).
  • the level of the liquid is determined independently of the thickness of the foam.
  • the level of a liquid or the liquid level means, for example, height of the liquid in a vessel.
  • the level of the liquid may be determined from the quantity of liquid present in the vessel in the absence of the foam i.e. prior to foam formation, and/or may be determined using a method that is largely independent of the presence or absence of the foam.
  • the level of the liquid may be determined by measurement of the mass of the content in a vessel as liquid (even though the mass includes the mass of the liquid and the mass of the foam, the mass of the foam is typically not significant compared to the mass of the liquid, and so the mass may be assumed to correlate with liquid level).
  • “Quantity” here may be based on any appropriate property that may be used to determine the level of the liquid within the vessel, such as mass, volume, depth, and from each of which a “level of the liquid” may be determined.
  • Thickness of the foam may be a relative measure, in comparison to the level of the liquid, or an absolute measure, e.g. the distance between the phase surface and a reflected signal receiver, for example. Thickness of the foam or the foam thickness means, for example, the difference between the level of a phase surface and the level of the liquid.
  • the level of the phase surface may be determined based on a time elapsed between emission of the signal and detection of the reflected signal. The level of the phase surface may therefore be determined based on the time of flight of an interrogating signal to be sent, and reflected from, the phase surface.
  • the signal may comprise electromagnetic radio waves, or radio waves.
  • the signal may be any signal suitable to provide the intended function, wherein the signal permits the recording of time of flight in order to determine the phase surface level. It will be apparent that some types of signals are appropriate for a given medium, while others are less appropriate for a given medium, e.g. the medium is transparent to, or an absorber of, that particular signal.
  • the medium should be a reflector of the chosen signal.
  • the signal may comprise radio waves in the range 26GHz – 170 GHz. Radio signals in the aforementioned range are resistant to degradation/attenuation from water droplets suspended in the head space (e.g. from sparging). [0016] The signal may be emitted towards and reflected from varying locations of the phase surface.
  • the level of the phase surface may be determined as an average, or a “high spot” (i.e., area of phase surface that is arranged closer to the receiver or is at a greater distance from the liquid level relative to other areas of the phase surface).
  • a “high spot” i.e., area of phase surface that is arranged closer to the receiver or is at a greater distance from the liquid level relative to other areas of the phase surface.
  • the acceptable thickness of the foam may vary across a phase surface, so a larger amount of foam may be permitted in regions of the phase surface remote from, e.g., filters or other elements of the vessel that are negatively impacted by excess foam.
  • the level of the liquid may be determined by volume determination and/or determination of a dimension associated with the liquid.
  • the volume may be predetermined (i.e., a known volume of liquid is added to the vessel of a given geometry), or directly determined via, e.g. liquid level sensor, capacitance sensor, guided wave radar and the like.
  • the volume of liquid may be indirectly determined, such as via measuring the mass of the fluid in the vessel and then converting to the liquid volume via density calculations and vessel geometry.
  • the method used to determine liquid volume may be varied during use of the vessel with some methods being more appropriate at differing stages, e.g., predetermined at the start of use of the vessel, with direct and/or indirect determination used at later time point(s).
  • the level of the liquid may be determined by volume determination associated with the liquid, and wherein the volume determination is based on a mass of the liquid.
  • the level of the phase surface and the level of the liquid may each be determined substantially simultaneously. In this way the thickness of the foam may be monitored in real time.
  • the dosage of an antifoaming additive may be controlled based on the measured thickness of the foam. Especially, it may be controlled by the comparison of the measured thickness of the foam to a threshold thickness of the foam. When the amount of foam surpasses the threshold thickness of the foam, the dosage of the antifoaming additive may be altered in order to ensure the amount of foam remains below the threshold. “Dosage” in this application may refer to any suitable measure or regimen for applying the antifoaming agent to the liquid in order to maintain the thickness of the foam within desired limits.
  • Dosage may therefore be varied by changing time intervals between additions of a constant quantity, or the quantity and/or concentration of the antifoaming additive in an addition may be changed and the time between dosage fixed, or a combination of the aforesaid.
  • the dosage may comprise no addition of the antifoaming additive when the measured thickness of the foam is below the threshold thickness of the foam, and addition of an aliquot (e.g. a constant quantity) of the antifoaming additive when the measured thickness of the foam is above the threshold thickness of the foam.
  • the dosage of the antifoaming additive may, at least in part, be determined by the deviation of the determined level from the threshold level.
  • the threshold level may, at least in part, be determined based upon an available head space in the vessel.
  • the vessel comprising liquid typically further includes a head space located above the liquid level and normally occupied by a gas.
  • the head space may vary according to the variation in the liquid level and the constant or maximum volume of the vessel. Accordingly, the acceptable thickness of the foam may vary depending on the available head space within the vessel at any given moment. A relatively higher liquid level may result in a lower amount of head space, and in turn, a lower acceptable thickness of the foam.
  • the threshold may account for the head space within the vessel and changes in the head space.
  • the threshold may vary according to the acceptable thickness of the foam within the vessel at any given moment.
  • the threshold level may, at least in part, be set at a specified level of the foam in the vessel relative to the level of the liquid.
  • the threshold level may, at least in part, be set at a rate of change of the thickness of the foam, with antifoaming agent added when a rapid increase of the thickness of the foam is detected within the vessel.
  • the dosage of the antifoaming additive may be controlled by adjusting the interval of adding the antifoaming additive and/or the amount of the antifoaming additive added in one instance. As discussed elsewhere, dosage may be varied by changing time intervals between additions of a constant quantity, or the quantity and/or concentration of an addition may be changed and the time between dosage fixed, or a combination of the aforesaid.
  • “instance” means a specific, individual, dose that is administered to the vessel. The instant dose may therefore be unique in the quantity of antifoaming agent provided relative to other instant doses which have previously, or will subsequently, be administered to the vessel.
  • the total dosage of the antifoam shall also consider the impact of the antifoam additives to the cell culture performance.
  • the vessel may be a bioreactor.
  • the vessel may be a bioreactor of a volume in the range of from about 1L to about 50,000L; however, as the skilled person will appreciate, the present method may be adapted for a bioreactor of any given size, shape, and dimensions.
  • the liquid may be a cell culture fluid comprising cell culture medium and cells.
  • the present method may be adapted (e.g. on the fly) to accommodate changing liquid properties, such as mass, density, propensity to form foam, degree of foam formation, and foam stability, all of which may change over the typical time-course of a cell culture process.
  • the method may be performed for a duration of a batch culture, a fed-batch culture, or a perfusion culture process.
  • the method may be repeated multiple times for the duration of the culture.
  • the method may be performed during the entire course of the culture, if desired, thereby ensuring the thickness of the foam is controlled within the tolerable level automatically.
  • the repetition may be prompted by addition or subtraction of an amount of liquid from the vessel and/or the elapsing of a period of time.
  • the method may be therefore performed, “on demand”, either as and when, on a whim, or at times where a rapid change in the thickness of the foam is expected (e.g. on the addition of an agent with known foam forming properties).
  • the method may be repeated substantially continuously for the duration of the culture.
  • a culture may have any suitable duration for the culture to complete or reach a desired end state. This duration may be minutes, hours, days, weeks, or months in length.
  • a signal with a refresh rate of the order of 5Hz may be used to provide 5 readings per second. Such a rate is suitably short as to capture rapid changes in the foam thickness. Therefore “continuously” in the context of this application means repeated interrogations of the foam thickness rather than a non-stop single interrogation of the foam thickness, which would provide an average foam thickness over a time course and obfuscate peaks in the foam thickness.
  • a method of producing a biologic from cells comprising: a) culturing cells in a cell culture fluid in a bioreactor; b) measuring the thickness of a foam within the bioreactor comprising the cell culture fluid; c) controlling the dosage of an antifoaming additive to be added to the cell culture fluid based on the measured thickness of the foam; and d) obtaining the biologic produced by the cultured cells; wherein the thickness of the foam in step b is determined based on the difference between the level of a phase surface and the level of the cell culture fluid; and wherein the phase surface is determined by a signal emitted towards and reflected from the phase surface.
  • the level of the phase surface may be determined based on a time elapsed between emission of the signal and detection of the reflected signal.
  • the signal may comprise electromagnetic radio waves.
  • the signal may comprise radio waves in the range 26GHz – 170 GHz., The signal may optionally comprise 80GHz radio waves. Radio signals in the aforementioned range are considered to be a compromise between a need for resistant to degradation/attenuation from water droplets suspended in the head space (e.g. from sparging) and a need to reduce the penetration through the foam which is inherent for a higher frequency radar signal.
  • the signal may be emitted towards and reflected from varying locations of the phase surface.
  • the level of the cell culture fluid may be determined by volume determination and/or determination of a dimension associated with the cell culture fluid.
  • the level of the cell culture fluid may be determined by volume determination associated with the cell culture fluid, and wherein the volume determination is based on a mass of the cell culture fluid.
  • the level of the phase surface and the level of the cell culture fluid may each be determined substantially simultaneously.
  • the dosage of an antifoaming additive may be controlled by the comparison of the measured thickness of the foam to a threshold thickness of the foam.
  • the dosage of the antifoaming additive may, at least in part, be determined by the deviation of the determined level from the threshold level.
  • the threshold level may, at least in part, be determined based upon an available head space in the vessel.
  • the dosage of the antifoaming additive may be controlled by adjusting the interval of adding the antifoaming additive and/or the amount of the antifoaming additive added in one time.
  • the bioreactor may have any suitable volume.
  • the bioreactor may have a volume in the range of from about 1L to about 50,000L.
  • the cell culture fluid may comprise cell culture medium and cells.
  • Steps b) and c) may be performed for a duration of a batch culture, a fed-batch culture, or a perfusion culture.
  • the steps b) and c) may be repeated multiple times for the duration of the culture. [0047] The repetition may be prompted by addition or subtraction of an amount of cell culture fluid from the vessel and/or the elapsing of a period of time. [0048] The steps b) and c) may be repeated substantially continuously for the duration of the culture. [0049] Step d) may be performed only at about the end of the duration of the culture. [0050] Step d) may comprise isolating the biologic from the cell culture fluid. [0051] The biologic may be at least one recombinant polypeptide and the cells may be or comprise eukaryotic cells comprising a polynucleotide encoding the recombinant polypeptide.
  • the at least one recombinant polypeptide may be selected from a fusion protein, an antibody, an antigen, an enzyme, or a vaccine.
  • an apparatus for controlling the thickness of the foam within a vessel comprising a liquid comprising: a vessel configured to comprise a liquid; phase surface level determining means, configured to determine the level of the phase surface, which comprises; an emitter, configured to emit a signal towards a phase surface; a receiver, configured to detect a reflected signal from the phase surface; liquid level determining means, configured to determine the level of the liquid in the vessel; and a processor, configured to; determine the thickness of a foam based on the difference between the level of the phase surface and the level of the liquid; and instruct the dosage of an antifoaming additive to be added to the liquid based on the determined thickness of the foam.
  • “vessel configured to comprise a liquid” means a vessel capable of containing and reserving a volume of liquid such that the vessel may be used to complete a relevant task, e.g. cell culture and/or biologic production.
  • the processor is typically arranged in communication with the emitter, receiver, liquid level determining means such that outputs from each may be received by the processor for determining the thickness of a foam based on the difference between the level of the phase surface and the level of the liquid.
  • the processor may be further arranged in communication with an output such that the processor is able to issue the instructions regarding the dosage of antifoaming additive.
  • the output may be any suitable so that changes in dosage of the antifoaming agent are affected.
  • the phase surface level determining means may determine the level of the phase surface based on a time elapsed between emission of the signal toward the phase surface and detection of the reflected signal. Accordingly, the apparatus may be capable of logging the time a signal is emitted and then received at the emitter and receiver respectively, such as either locally at the respective emitter and receiver, and then such times are passed to the processor, or by the processor instructing emission and awaiting detection via the emitter and receiver.
  • the emitter and/or receiver may be arranged substantially perpendicular to the phase surface of the liquid in the vessel. In this way the reflected signal is maximized.
  • the emitter and/or receiver may be arranged above the top of the vessel.
  • the emitter and/or receiver may be arranged on an outside of the vessel to respectively emit or detect signal to an inside of the vessel.
  • the emitter and/or receiver may at least partially pass through a wall of the vessel. Locating the emitter and/or receiver outside the vessel is advantageous in facilitating ease of inspection, testing, and maintenance if the receiver and/or emitter. The most important advantage for the radar mounting outside of the bioreactor is that it is the outside of sterile boundary of a bioreactor so no cleaning and sterilization are needed for the next use.
  • the emitter and/or receiver may be arranged on an inside of the vessel to respectively emit or detect signal within the vessel.
  • Locating the emitter and/or receiver inside the vessel is advantageous in that the method may be used with vessels formed of materials that are opaque to the emitted signal, as the signal need not traverse the opaque vessel and instead may be detected by the internal receiver.
  • the emitter may be configured to emit any of: electromagnetic radio waves; or radio waves.
  • the emitter may be configured to emit radio waves in the range 26GHz – 170 GHz.
  • the liquid level determining means may be selected from a load cell, a scales, a ranging apparatus, and a guided wave radar.
  • the liquid level determining means may determine the liquid level directly (in the case of ranging apparatus or guided wave radar) or indirectly via density and/or volume calculations in the case of a load cell or weighing scales.
  • the liquid level determining means may include means to determine the mass of the liquid (such as, but not limited to, the load cell, or scales), thereby allowing the volume to be calculated using the density of the liquid and vessel geometry.
  • the vessel may be a bioreactor.
  • the bioreactor may have a volume in the range of from about 1L to about 50,000L.
  • the vessel may formed, at least in part, of a signal-transparent material, and wherein the emitter and/or receiver is arranged to respectively emit or detect a signal through the signal- transparent material.
  • the top of the vessel may be formed of a signal-transparent or signal-transparent material.
  • the vessel may be advantageously formed of a material that is translucent or transparent to the signal, an appropriate material may be selected depending on whether the signal comprises electromagnetic radio waves; or radio waves such that the material is translucent/transparent to the signal.
  • Translucent/transparent here means that the signal is attenuated and/or degraded little or not at all on passing through the material.
  • the vessel may be formed, at least in part, of a radio-opaque material.
  • the vessel may be provided with a window formed of a signal transparent or translucent material with the rest of the vessel formed of a signal opaque material. This may be for performance, economic, structural, or other reasons where the benefits conferred by the signal opaque material warrant its use rather than forming the entire vessel from a signal transparent or translucent material.
  • the vessel may be formed, at least in part, of stainless steel; glass; ceramic; and/or a plastic film.
  • the vessel may comprise at least a partial stainless steel shell and a plastic film liner.
  • the processor may control the dosage of an antifoaming additive by comparing the determined thickness of the foam to a threshold thickness of the foam.
  • the processor controls the dosage of an antifoaming additive by determining the deviation of the determined level from the threshold level.
  • the processor may be configured to determine the thickness of the foam and instruct the dosage of an antifoaming additive repeatedly and/or continuously during a cell culture duration.
  • the processor may be configured to instruct the dosage of an aliquot (e.g. a constant quantity) of the antifoaming additive when the measured thickness of the foam is above the threshold thickness of the foam.
  • the apparatus may further comprises an antifoaming additive injector configured to automatically inject the antifoaming additive into the vessel when the processor instructs the addition of the antifoaming additive.
  • the antifoaming additive injector is arranged in communication with the processor such that the antifoaming additive injector acts on instruction from the processor.
  • Figure 1 shows a cross section of a vessel according to a first embodiment
  • Figure 2 shows a second cross section of a vessel according to the first embodiment
  • Figure 3 shows a third cross section of a vessel according to the first embodiment
  • Figure 4 shows a cross section of a vessel according to a second embodiment
  • Figure 5 is a plot showing the change in the phase surface level, the liquid level, and load cell output plotted with time
  • Figure 6 is a plot showing the measured foam thickness with batch feeds plotted with time
  • Figure 7 is a plot showing the measured foam thickness, various thresholds, and antifoam addition timing with time.
  • Figure 8 is a diagram of an example cell culture system comprising an automatic antifoam monitoring and addition system.
  • Figure 9 is a diagram of a cell culture run that depicts foam thickness (black line) in inches (Y-axis left) in response to various threshold foam thickness settings (gray dashed line) during cell culture and in response to additions of antifoam when the antifoam addition pump is on (vertical dotted gray lines at X-axis) at various time points (X axis), and the weight of the contents of the cell culture vessel as cell culture progresses (black dashed line) (Y axis right, kilograms).
  • the present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • cell culture fluid refers to a fluid that may be used for culturing cells, such as (an aqueous) fluid comprising a “cell culture medium” or “culture medium”.
  • cell culture medium and “culture medium” refer to a nutrient solution used for growing mammalian cells that typically provides at least one component from one or more of the following categories: 1) an energy source, usually in the form of a carbohydrate such as glucose; 2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; 3) vitamins and/or other organic compounds required at low concentrations; 4) free fatty acids; and 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • an energy source usually in the form of a carbohydrate such as glucose
  • all essential amino acids and usually the basic set of twenty amino acids plus cysteine
  • vitamins and/or other organic compounds required at low concentrations 4) free fatty acids; and 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.
  • the nutrient solution can optionally be supplemented with one or more components from any of the following categories: 1) hormones and other growth factors as, for example, insulin, transferrin, and epidermal growth factor; 2) salts and buffers as, for example, calcium, magnesium, and phosphate; 3) nucleosides and bases such as, for example, adenosine, thymidine, and hypoxanthine; and 4) protein and tissue hydrolysates.
  • “Culturing” a cell refers to contacting a cell with a cell culture medium under conditions suitable to the survival and/or growth and/or proliferation of the cell.
  • Batch culture refers to a culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing bioreactor at the start of the culturing process.
  • “Fed-batch cell culture,” as used herein refers to a batch culture wherein the cells and culture medium are supplied to the culturing bioreactor initially, and additional culture nutrients are fed, continuously or in discrete increments, to the culture during the culturing process, with or without periodic cell and/or product harvest before termination of culture.
  • Perfusion culture is a culture by which the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers, etc., and the culture medium is continuously, step-wise or intermittently introduced (or any combination of these) and removed from the culturing bioreactor.
  • the term “cells” refers to animal cells (e.g. mammalian cells), bacteria cells (e.g. E. Coli cells), fungal cells (e.g. yeast cells), cultured cells, host cells, recombinant cells and recombinant host cells.
  • Such cells are generally cell lines obtained or derived from mammalian tissues or fungi which are able to grow and survive when placed in media containing appropriate nutrients and/or growth factors.
  • the term “cell line” as used herein includes reference to a culture of (for example) eukaryotic cells that can be propagated repeatedly. The eukaryotic cells of the cell line may be selected from any cell as defined herein.
  • the terms “host cell,” “host cell line” and “host cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells.
  • host cells may also have been modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell products (e.g., endogenous virus-like particles or endogenous host cell proteins).
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny does not need to be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • exogenous nucleic acid e.g., by transfection
  • host cell e.g., by transfection
  • host cell line e.g., by transfection
  • host cell culture may also refer to such recombinant cells and their progeny.
  • the recombinant product expressed by such cells may be a recombinant protein, a recombinant viral particle, or a recombinant viral vector.
  • the term “mammalian host cell” or “mammalian cell” refers to cell lines derived from mammals that are capable of growth and survival when placed in either monolayer culture or in suspension culture in a medium containing the appropriate nutrients and growth factors. The necessary growth factors for a particular cell line are readily determined empirically without undue experimentation, as described for example in Mammalian Cell Culture (Mather, J. P. ed., Plenum Press, N.Y.1984), and Barnes and Sato, (1980) Cell, 22:649.
  • the cells are capable of expressing and secreting large quantities of a particular protein of interest into the culture medium.
  • suitable mammalian host cells within the context of the present disclosure can include Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:42161980); dp12.CHO cells (EP 307,247 published 15 Mar. 1989); CHO-K1 (ATCC, CCL-61); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol.
  • Chinese hamster ovary cells/-DHFR CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:42161980
  • dp12.CHO cells EP 307,247 published 15 Mar. 1989
  • CHO-K1 ATCC, CCL-61
  • baby hamster kidney cells BHK, ATCC CCL 10
  • mouse sertoli cells TM4, Mather,
  • exemplary mammalian cells include Chinese hamster ovary cells (CHO).
  • the cells may comprise a polynucleotide that encodes a polypeptide.
  • biological or “biologics” as used herein refers to molecules that are produced by cells, such as such as proteins, glycans, or lipids.
  • the biologics may be biologically active molecules.
  • a biologic may be a recombinant polypeptide, for example a recombinant polypeptide selected from a fusion protein, an antibody, an antigen, an enzyme, and a vaccine.
  • Biologics may be excreted by the cells into the cell culture fluid, which may facilitate isolation of the biologics.
  • expression or “expresses” are used herein to refer to transcription and translation occurring within a host cell.
  • the level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell. For example, mRNA transcribed from a product gene is desirably quantitated by northern hybridization.
  • Protein encoded by a product gene can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein.
  • polypeptide refers generally to peptides and proteins having more than about ten amino acids.
  • the polypeptides can be homologous to the host cell, or preferably, can be exogenous, meaning that they are heterologous, i.e., foreign, to the host cell being utilized, such as a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell.
  • mammalian polypeptides polypeptides that were originally derived from a mammalian organism
  • proteins is meant to refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure.
  • the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD.
  • proteins encompassed within the definition herein include host cell proteins as well as all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds.
  • the term “glycoprotein” refers to a protein which contains an oligosaccharide chain covalently attached to amino acid side-chains.
  • the oligosaccharide(s) may be attached to the protein in a co-translational or post-translational modification, during a process known as glycosylation.
  • Exemplary glycoproteins include antibodies, which typically have an N-linked oligosaccharide on each heavy chain.
  • antibody is used herein in the broadest sense and encompasses various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence, including multimers of such pairings), multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen- binding activity.
  • a therapeutic antibody is an antibody that may be used in the treatment of a disease.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • Titer refers to the total amount of recombinantly expressed antibody produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or liter of medium (mg/ml or mg/L).
  • titer is expressed in grams of antibody per liter of medium (g/L). Titer can be expressed or assessed in terms of a relative measurement, such as a percentage increase in titer as compared obtaining the protein product under different culture conditions.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies in accordance with the presently disclosed subject matter can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non- human antigen-binding residues.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human complementarity determining regions (CDRs) and amino acid residues from human framework regions (FRs).
  • CDRs non-human complementarity determining regions
  • FRs human framework regions
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • the term “enzyme” as used herein refers to a protein or polypeptide (such as a recombinant polypeptide) that acts as a biological catalyst.
  • the terms “recombinant protein” and “recombinant polypeptide” refer generally to peptides and proteins, including antibodies, that are encoded by a nucleic acid that is “heterologous,” i.e., foreign to the host cell being utilized, such as a nucleic acid encoding a human antibody that is introduced into a non-human host cell.
  • the vessel is capable of holding a liquid 102 within an interior of the vessel 101.
  • the vessel 101 is formed, at least in part, of stainless steel; glass; ceramic; and/or a plastic film or any other suitable materials as appropriate to manufacturing process.
  • the liquid 102 may be added or removed from the vessel 101 at will (for example from one or more inlet or outlet ports (not illustrated), and the quantity of liquid 102 can be controlled or predetermined.
  • quantity may be specified in any suitable units, such as, but not limited to mass or volume.
  • the vessel may be sealed or gated (by way of a valve) from the external environment (i.e. the vessel 101 comprises a cavity) or the vessel may be open to the environment, provided the vessel is still configured to hold liquid.
  • the vessel may also be configured to provide sparging (not shown) of held liquid 102 with air, oxygen, or any other suitable gas and / or the vessel may comprise an agitator (not shown) for mixing any held liquid 102.
  • the apparatus 100 is further equipped with phase surface level determining means.
  • the phase surface level determining means comprising an emitter 103 and a receiver 104, may be attached on the vessel 101, or be separate from the vessel 101. Where the phase surface level determining means is separate from the vessel 101, the phase surface level determining means may still be arranged in contact with the vessel (if not attached), or may be spaced apart from the vessel, provided the spacing does not prevent the phase surface level determining means performing its intended function.
  • the phase surface level determining means provides the function of detecting the presence and location of the transition between two phases as matter, such as liquid – gas or foam – gas in the present application.
  • the phase surface level determining means comprises an emitter 103 and a receiver 104.
  • the emitter 103 is configured to emit a signal towards the interior of the vessel 101, such that the location of the phase surface 105 lies along the path the signal travels.
  • the signal emitted may be as per any signal discussed in this application, such as signal radio waves in the range 26GHz – 170 GHz.
  • the phase surface may act as a reflector for the signal. The signal, when it encounters the phase surface, is reflected.
  • the whole of the emitted signal may not be reflected.
  • a partial signal may be reflected by the phase surface.
  • the amount of signal reflected may vary depending on the angle of incidence of the signal with the phase surface, or the nature of the phase surface (i.e. particular phases of matter and physical/chemical properties of the materials forming the phase surface at any given time).
  • the receiver 104 is configured to detect the signal, or the portion of the signal that is reflected back towards the receiver 104. It is noted that the portion of the signal that is reflected back towards the receiver may not be the totality of the reflected signal.
  • the phase surface 105 may scatter a portion of the reflected signal, such that the path of parts of the reflected signal are not co-incident with the receiver 104.
  • the receiver may be arranged adjacent with the emitter, the receiver and emitter may form part of a single unit. Alternatively, the emitter and receiver may be separate units arranged adjacent one another. Further alternatively, the emitter and receiver may be arranged in separate positions, spaced apart from one another provided the receiver is arranged such that the receiver is capable of receiving a signal emitted from the emitter following reflection from the phase surface.
  • the vessel 101 comprises a wall configured to hold the liquid 102. As the emitter 103 and receiver 104 are arranged external to the vessel, the vessel wall should be formed of a signal transparent material, at least proximate to where the emitter 103 and receiver 104 are situated, in order to allow the signal to pass through.
  • a liquid level determining means 106 is also provided.
  • the liquid level determining means may be any as described in this application. As previously discussed the level of the liquid may be determined directly or indirectly.
  • Direct methods include ranging apparatus and/or guided wave radar. Indirect methods include load cell or a scales.
  • the vessel 101 in the figures is shown arranged on a scales as the liquid level determining means 106.
  • a processor (not shown) is provided in communication with the liquid level determining means 106, the emitter 103, and the receiver 104 and arranged to receive signals from those elements.
  • the apparatus 100 is further provided with an injector 107 which is arranged to release a pre-determined dosage of antifoaming agent into the vessel.
  • the vessel 101 comprises a liquid 102 and a process is underway in the vessel.
  • a foam 200 has formed on top of the liquid 102, typically between the top of the liquid and a head space 201 of the vessel 101.
  • the head space 201 is a working area of the vessel 101 which is not presently occupied by the liquid and/or foam.
  • the head space 201 is the space in the vessel which is not occupied by the liquid or a foam.
  • the head space 201 will change in dimensions depending on changes in volume of the liquid and/or foam (e.g. by addition or expansion of the liquid volume).
  • the head space 201 is typically occupied by a gas.
  • the foam 200 comprises a liquid contact surface 205 between the liquid 102 and the foam 200, and a phase surface 202 between the foam 200 and the head space 201.
  • the emitter 103 is shown emitting a signal 203 towards the foam-head space phase surface 202.
  • the emitted signal 203 is reflected on the phase surface, and at least a portion of the reflected signal 204 is detected by the receiver 104.
  • a time of flight of the signal is calculated by the time it takes from the emission of the emitted signal 203 to be detected as a reflected signal 204 at the receiver 104.
  • the distance of the foam-head space surface relative to the receiver 104 may therefore be determined with short durations indicating that the foam-head space phase surface 202 is relatively closer to the receiver 204 than detections having longer durations.
  • the liquid level determining means 106 may, at the same time as the detection of the signal, provide a value for the liquid level in the vessel, absent foam, which may be used to determine the volume of the vessel that would be occupied by the liquid without a foam, with the remainder of the vessel interior being available head space 201.
  • the available head space 201 may then be compared to the distance of the foam-head space surface relative to the receiver 104 and thereby determine the thickness of the foam.
  • the processor may instruct the dosage of an antifoaming additive to be altered.
  • the dosage may be determined as previously described.
  • Antifoaming additive may be provided per the calculated dosage via the injector 107 into the vessel 101 to suppress the foam.
  • Monitoring of the thickness of the foam via detection of the foam-head space phase surface and liquid level determining means may then continue, repeated continuously, until the foam forming process within the vessel ends. In this way the thickness of the foam within the vessel may be automatically controlled without external input. The method is not reliant upon viewing the contents of the vessel and is capable of operating with variations in the liquid quantity.
  • the acceptable thickness of the foam may be defined as a threshold, which once surpassed, triggers the dosage of the antifoaming agent to be altered.
  • the alteration of the dosage comprises no addition of the antifoaming agent when the measured thickness of the foam is below the threshold, and addition of an aliquot (e.g. a constant quantity) of the antifoaming agent when the measured thickness of the foam is above the threshold.
  • an aliquot e.g. a constant quantity
  • the emitted signal 303 and the detected signal 304 are shown interrogating the foam- head space phase surface at a location distant from the emitter 103 and the receiver 104. It may be that the acceptable thickness of the foam within the vessel may be non-uniform, with areas of the head space within the vessel tolerating a larger amount of foam compared to other areas (e.g. those areas near filters for example). [0126] Interrogating multiple locations across the foam-head space phase surface also allows for the identification of areas of increased foam thickness relative to other areas as foam formation within the vessel may be non-uniform.
  • FIG. 4 depicts apparatus 400 similar to that of figures 1 and 2, having the same features and properties as previously described. Like reference numerals have been omitted so as to not unnecessarily obscure the figure.
  • the apparatus 400 differs from the apparatus of earlier figures in that the emitter 403 and the receiver 404 are arranged within the head space of the vessel rather than outside the vessel as shown in figures 1, 2, and 3. Other arrangements are possible as described elsewhere. As the emitter 403 and the receiver 404 are arranged within the head space, the vessel need not be formed of a signal transparent material, and may instead be formed of a signal opaque material.
  • ADDITIONAL EMBODIMENTS [0128] Further embodiments of the invention are described in the below numbered clauses: Clause 1.
  • a method of monitoring and controlling the foam within a vessel comprising a liquid comprising: a) measuring the thickness of the foam within a vessel comprising a liquid; and b) controlling the dosage of an antifoaming additive to be added to the liquid based on the measured thickness of the foam; wherein the thickness of the foam in step a is determined based on the difference between the level of a phase surface and the level of the liquid; and wherein the level of the phase surface is determined by a signal emitted towards and reflected from the phase surface.
  • the dosage of the antifoaming additive is controlled by adjusting the interval of adding the antifoaming additive and/or the amount of the antifoaming additive added in one instance.
  • the vessel is a bioreactor of a volume in the range of from about 1L to about 50,000L.
  • the liquid is a cell culture fluid comprising cell culture medium and cells.
  • Clause 17 The method of any previous clause, wherein the method is performed for a duration of a batch culture, a fed-batch culture, or a perfusion culture.
  • Clause 18 The method of any previous clause, wherein the method is repeated multiple times for the duration of a culture. Clause 19.
  • a method of producing a biologic from cells comprising: a) culturing the cells in a cell culture fluid in a bioreactor; b) measuring the thickness of a foam within the bioreactor comprising the cell culture fluid; c) controlling the dosage of an antifoaming additive to be added to the cell culture fluid based on the measured thickness of the foam; and d) obtaining the biologic produced by the cultured cells; wherein the thickness of the foam in step b is determined based on the difference between the level of a phase surface and the level of the cell culture fluid; and wherein the phase surface is determined by a signal emitted towards and reflected from the phase surface.
  • Claims 22 The method of clause 21, wherein the phase surface is the surface between foam and headspace gas in the vessel.
  • Clause 23 The method of clause 21, wherein the level of the phase surface is determined based on a time elapsed between emission of the signal and detection of the reflected signal.
  • Clause 24 The method of any of clauses 21 to 23, wherein the signal comprises any of: electromagnetic radio waves; or radio waves.
  • Clause 25 The method of any one of clauses 21 to 24, wherein the signal comprises radio waves in the range 26GHz – 170 GHz, and optionally wherein the signal comprises 80GHz radio waves.
  • Clause 26 The method of any one of clauses 21 to 25, wherein the signal is emitted towards and reflected from varying locations of the phase surface.
  • Clause 35 The method of any one of clauses 21 to 34, wherein the cell culture fluid comprises cell culture medium and cells.
  • Clause 36 The method of any one of clauses 21 to 35, wherein the steps b) and c) are performed for a duration of a batch culture, a fed-batch culture, or a perfusion culture.
  • Clause 37 The method of any one of clauses 21 to 36, wherein the steps b) and c) are repeated multiple times for the duration of the culture.
  • Clause 38 The method of clause 37, wherein the repetition is prompted by addition or subtraction of an amount of cell culture fluid from the vessel and/or the elapsing of a period of time.
  • Apparatus for controlling the thickness of the foam within a vessel comprising a liquid comprising: a vessel configured to comprise a liquid; phase surface level determining means, configured to determine the level of the phase surface, which comprises; an emitter, configured to emit a signal towards a phase surface; a receiver, configured to detect a reflected signal from the phase surface; liquid level determining means, configured to determine the level of the liquid in the vessel; and a processor, configured to; determine the thickness of a foam based on the difference between the level of the phase surface and the level of the liquid; and instruct the dosage of an antifoaming additive to be added to the liquid based on the determined thickness of the foam.
  • Clause 45 The apparatus of clause 44, wherein the phase surface level determining means determines the level of the phase surface based on a time elapsed between emission of the signal toward the phase surface and detection of the reflected signal.
  • Clause 46 The apparatus of clauses 44 or 45, wherein the emitter and/or receiver are arranged substantially perpendicular to the phase surface of the liquid in the vessel.
  • Clause 47 The apparatus of any one of clauses 44 to 46, wherein the emitter and/or receiver are arranged above the top of the vessel.
  • Clause 48 The apparatus of any one of clauses 44 to 47, wherein the emitter and/or receiver are arranged on an outside of the vessel to respectively emit or detect signal to an inside of the vessel.
  • Clause 62 The apparatus of any one of clauses 44 to 61, wherein the apparatus further comprises an antifoaming additive injector configured to automatically inject the antifoaming additive into the vessel when the processor instructs the addition of the antifoaming additive.
  • Clause 63 Use of an apparatus of any of clauses 44 to 62 for controlling the thickness of the foam in a vessel, optionally wherein the vessel is a bioreactor.
  • Clause 64 Use of an apparatus of any of clauses 44 to 62 for culturing a cell line. Clause 65.
  • the biologic is at least one recombinant polypeptide and the cell line is a eukaryotic cell line comprising a polynucleotide encoding the recombinant polypeptide; optionally wherein the recombinant polypeptide is a fusion protein, an antibody, an antigen, an enzyme, or a vaccine.
  • Clause 68 The use of clause 66 or clause 67, further comprising isolating the biologic.
  • Any antifoaming additive or agent suitable for mammalian or prokaryotic cell culture may be used for the methods and other embodiments disclosed herein.
  • the antifoaming additive or agent is a silicone based antifoam agent, comprising for example, one or more of a simethicone; a polydimethyl siloxane; and/or a silicon dioxide.
  • the antifoam additive or agent is LIVEOTM (DuPontTM).
  • the radar comprised an emitter and a receiver, was positioned proximate to the top of the bioreactor and was configured to emit and receive a signal to enable determination of the level of a phase surface in the bioreactor.
  • the load cell was used to measure the mass of the cell culture in a bioreactor, which was converted to the level of the liquid, using a previously determined calibration, based on the volume and geometry of the bioreactor.
  • the bioreactor further comprised inlets for separate addition of cell culture feed and antifoam additive.
  • the bioreactor also comprised means for gas sparging.
  • the bioreactor was in communication with a processor.
  • the processor was configured to determine the liquid level from the output of the load cell, determine the thickness of the foam from the difference between the level of the phase surface and the level of the liquid, and automatically add antifoam additive based on a pre-planned schedule.
  • An experiment was performed to confirm the ability of the apparatus to continuously measure the thickness of the foam in the bioreactor over an extended period of time. Results are provided in Figures 5 and 6.
  • Figure 5 is a plot of the phase surface level, the load cell output, and the converted liquid level from the load cell output over time in an implementation of the disclosure.
  • the x-axis shows the passage of time over the course of 10 days.
  • the output of the load cell is shown with changes during a manufacturing process progresses.
  • the units of the load cell output can be any suitable as described herein, provided the output of the load cell can be used to determine the liquid level, absent foam.
  • the changes in load cell output could be, by way of non-limitative example, changes in the liquid volume either through addition/subtraction of liquid and/or growth of a product in the vessel in a bioprocess.
  • the liquid level, expressed as percent height of the vessel is calculated from the load cell output in Figure 5. As can be seen (and as would be expected), the liquid level closely follows the load cell output due to its cylindrical shape of the vessel in this example. Using a percentage as a unit of the vessel for levels of liquid and phase surface provides an advantage to make the calculation in a processor for the foam thickness more easily transferrable to other vessels.
  • the phase surface level measured by the radar is expressed as percent height of the vessel.
  • the liquid level converted from a load cell is also expressed as percent height of the vessel.
  • Subtracting the percent of the liquid level occupied in the vessel from the percent of the phase surface level provides the percent of the foam thickness in terms of vessel height, which is then converted to the actual foam thickness in inch in this case at any given moment as shown in Figure 6.
  • the foam thickness was determined using the techniques as hereinbefore described and plotted. When the foam was generated in the bioreactor vessel, the phase surface level is greater than the liquid level, and shows the foam thickness in inch is greater than 0. When there was no foam in the bioreactor, the foam thickness is zero.
  • Figure 6 shows thickness of the foam in circled dots determined by the measurements of the phase surface level and the liquid level displayed in Figure 5.
  • the x-axis shows the passage of time over the course of 10 days. Periodic batch feeds (dashed line) were made in the process, three times in this process. Foam was produced as the manufacturing process proceeded (e.g. from gas production and/or sparging). An antifoaming additive was added to the liquid over the 10 days by predetermined schedule. As shown in Figure 6, the apparatus continuously measured the thickness of the foam in the bioreactor over an extended period of time.
  • Example 2 Control of the dosage of an antifoaming additive based on the foam thickness
  • Figure 7 shows automatic antifoam control using the foam thickness measurement compared to a threshold thickness.
  • the foam thickness in the vessel was measured in the same manner as Example 1 and compared with a threshold thickness.
  • the threshold thickness (dotted line on horizontal axis) was initially set to 10 inches and then switched to 3 inches.
  • the dosage was set to add 20 g antifoam additive after 45 seconds from the end of the previous antifoam additive addition if the foam thickness was over the threshold thickness.
  • the threshold thickness was set to 10 inches, there was no antifoam additive addition because the foam thickness was always under the threshold value.
  • the first four doses were made following the dosage plan because the foam thickness was over the threshold thickness.
  • the threshold may be set in more than one way.
  • the threshold may be set at a specified level of the foam in the vessel relative to the level of the liquid at that time.
  • the threshold could be set at a rate of change of the thickness of the foam, with antifoaming agent added when a rapid increase of the thickness of the foam is detected within the vessel.
  • Figure 7 shows the successful automatic management of foam within a vessel using the disclosed methods. This provides important advantages (e.g.
  • FIG. 8 depicts an example automatic anti-foam control bioreactor cell culture system.
  • a 2000L bioreactor was loaded with 1200L CHO cell culture medium and inoculated from a standard cell culture seed train.
  • the bioreactor was equipped with load cells that detect and report on the mass of the cell culture in a bioreactor, which was converted to the level of the liquid based on the volume and geometry of the bioreactor.
  • the load cells can be replaced by using a guide-wave radar for a direct measurement of liquid height.
  • the bioreactor further comprised an 80 GHz radar positioned proximate to the top of the bioreactor.
  • the radar was configured to emit and receive a signal to enable determination of the level of a phase surface in the bioreactor.
  • an over-air radar was used to measure liquid or foam height, but the system can use a guided wave radar instead or in addition.
  • gases e.g., oxygen
  • the bioreactor was connected to an antifoam addition pump, which added an amount of liquid antifoam agent (e.g., LiveoTM (DuPontTM), a silicone based antifoam agent, comprising for example, one or more of a simethicone; a polydimethyl siloxane; and/or a silicon dioxide) into the bioreactor when the foam reaches a predetermined height.
  • liquid antifoam agent e.g., LiveoTM (DuPontTM
  • silicone based antifoam agent comprising for example, one or more of a simethicone; a polydimethyl siloxane; and/or a silicon dioxide
  • dose of antifoam was calculated for a 2000 L bioreactor; smaller or larger bioreactors would use accordingly less or more antifoam agent per dose.
  • the activity of the antifoam addition pump was controlled by a Foam Thickness Monitoring Module and an Antifoam Addition Control Module,
  • the Foam Thickness Monitoring Module received liquid height and foam height information from the radar to calculate foam thickness.
  • the Antifoam Addition Control Module directed the antifoam addition pump to switch on, dispensing a dose of antifoam into the cell culture medium.
  • the Antifoam Addition Control Module allowed the user to set a threshold acceptable foam height (Foam Thickness Threshold), amount of antifoam to add per dose, amount of time to delay addition of antifoam, etc.
  • Figure 9 depicts performance of the antifoam system during cell culture.
  • the X-axis indicates time points of culture after initial inoculation with seed train cell culture. (Note the settling seen at about time point 326 is likely due to foam settling without antifoam addition, and the settling seen at time point 456 is due to a manual foam addition.)
  • the left Y-axis indicates inches of foam in the bioreactor, and the right Y-axis indicates the bioreactor weight in kilograms.
  • the stepped line rising to the right is the weight of the bioreactor upon addition of cell culture medium, seed train cell culture, cell culture medium feedings, and inputs of antifoam.
  • the line at the bottom of the figure indicates times the antifoam pump is on when there is a peak (short vertical line).
  • the stepped line generally descending from left to right is the user-set foam thickness threshold.
  • the jagged line is the actual measured foam thickness during the course of cell culture. This line indicates a rise in foam thickness during cell culture and sparging, with a sharp drop at and just after the point at which the antifoam addition pump is on.

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Abstract

L'invention concerne un procédé de surveillance et de régulation de la mousse à l'intérieur d'un récipient comprenant un liquide. Le procédé comprend la mesure de l'épaisseur de la mousse à l'intérieur d'un récipient comprenant un liquide et la régulation du dosage d'un additif antimousse à ajouter au liquide en fonction de l'épaisseur mesurée de la mousse. L'épaisseur de la mousse est déterminée en fonction de la différence entre le niveau d'une surface de phase et le niveau du liquide. Le niveau de la surface de phase est déterminé par un signal émis vers la surface de phase et réfléchi par celle-ci. L'invention concerne également un appareil permettant de mettre en œuvre le procédé, ainsi qu'un autre procédé de production de produits biologiques à partir de cellules.
PCT/US2025/025130 2024-04-19 2025-04-17 Procédé de surveillance et de régulation de la mousse à l'intérieur d'un récipient Pending WO2025221977A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0307247A2 (fr) 1987-09-11 1989-03-15 Genentech, Inc. Méthode pour cultiver des cellules recombinantes
WO2020081128A1 (fr) * 2018-10-16 2020-04-23 Sartorius Stedim North America Inc. Atténuation de mousse dans des bioréacteurs à l'aide d'ultrasons
US20210123010A1 (en) * 2016-08-31 2021-04-29 Ge Healthcare Bio-Sciences Ab Detection of foam levels
US20210355427A1 (en) 2019-03-28 2021-11-18 Emd Millipore Corporation Use of vision systems in biomanufacturing processes
EP3458830B1 (fr) * 2016-05-17 2023-04-05 Solenis Technologies, L.P. Canon à mousse à flux latéral et son système de commande
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Publication number Priority date Publication date Assignee Title
EP0307247A2 (fr) 1987-09-11 1989-03-15 Genentech, Inc. Méthode pour cultiver des cellules recombinantes
EP3458830B1 (fr) * 2016-05-17 2023-04-05 Solenis Technologies, L.P. Canon à mousse à flux latéral et son système de commande
US20210123010A1 (en) * 2016-08-31 2021-04-29 Ge Healthcare Bio-Sciences Ab Detection of foam levels
WO2020081128A1 (fr) * 2018-10-16 2020-04-23 Sartorius Stedim North America Inc. Atténuation de mousse dans des bioréacteurs à l'aide d'ultrasons
US20210355427A1 (en) 2019-03-28 2021-11-18 Emd Millipore Corporation Use of vision systems in biomanufacturing processes
US20240084339A1 (en) * 2021-02-04 2024-03-14 Kaneka Corporation Culture device and use thereof

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"Mammalian Cell Culture", 1984, PLENUM PRESS
BARNESSATO, CELL, vol. 22, 1980, pages 649
CHO, URLAUBCHASIN, PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216
HOLLIGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
SAM BROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS, pages: 1 - 88

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