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WO2025141180A1 - Unité de distribution de substances nitritives pour bioréacteur - Google Patents

Unité de distribution de substances nitritives pour bioréacteur Download PDF

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Publication number
WO2025141180A1
WO2025141180A1 PCT/EP2024/088600 EP2024088600W WO2025141180A1 WO 2025141180 A1 WO2025141180 A1 WO 2025141180A1 EP 2024088600 W EP2024088600 W EP 2024088600W WO 2025141180 A1 WO2025141180 A1 WO 2025141180A1
Authority
WO
WIPO (PCT)
Prior art keywords
media
vessel
gas exchange
bioreactor
supply unit
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/EP2024/088600
Other languages
English (en)
Inventor
Nicolaas Willem DE JONGH
Remy Philippe T KUSTERS
Mejdi NCIRI
Victor Claude Léon SAYOUS
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.)
Supreme
Original Assignee
Supreme
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 Supreme filed Critical Supreme
Publication of WO2025141180A1 publication Critical patent/WO2025141180A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate

Definitions

  • the present invention relates to the field of cell culture systems and their optimization.
  • the invention relates to the field of advanced bioreactor technology, aimed at enhancing the efficiency of cell culture processes.
  • This invention can provide a cell culture system for industrial scale production, offering high-density cultivation and efficient nutrient distribution for optimal growth and productivity.
  • the invention aims to overcome the disadvantages of the prior art.
  • the invention proposes a nutrient supply unit for a bioreactor comprising a vessel, preferably for cell culture, the nutrient supply unit comprising: o a gas exchange device, the gas exchange device comprising a gas exchange surface and being configured to introduce a gas into the vessel, preferably without inducing shear stress in the vessel; o a media distribution device, the media distribution device being configured to introduce a culture media into the vessel; and o a media collection device, the media collection device being configured to remove the culture media from the vessel; said gas exchange device, media distribution device and media collection device, being arranged to induce a flow of media at the gas exchange surface.
  • Such a nutrient supply unit allows an enhancement of the mixing and oxygen transfer in a bioreactor. This improvement is crucial for cell growth and sustainability, as it ensures the cells receive the necessary nutrients and oxygen uniformly.
  • This solution is designed to increase efficiency without increasing shear stress, which is necessary for maintaining cell integrity at high scale.
  • a key advantage of this invention is its ability to minimize shear stress during the cell culture process. Excessive shear stress can damage delicate cell structures, impeding growth and productivity.
  • the technology employed in this invention allows for gentle yet effective mixing, ensuring cell viability and integrity are maintained, which is essential for high-quality cell-based product development.
  • the invention can simplify the maintenance process of bioreactors, which is often a challenging and time-consuming task.
  • this invention provides an effective solution in particular for industrial-scale bioreactors.
  • the present invention can be used to reduce production costs particularly in applications requiring large volumes of culture media, such as the production of cultivated meat or large-scale vaccine manufacturing.
  • the nutrient supply unit can optionally include one or more of the following characteristics alone or in combination: it is adapted to protrude within the vessel of the bioreactor. This can improve the mixing and oxygen transfer in a bioreactor.
  • the nutrient supply unit comprises a removable connector on an extremity. This can be particularly relevant when considering the combination of enhancing the mixing and oxygen transfer and enabling easier maintenance.
  • the nutrient supply unit is adapted to protrude within the vessel of the bioreactor.
  • it can protrude at least in part.
  • most or all the gas exchange surface protrudes within the vessel of the bioreactor. This can enhance the mixing and oxygen transfer.
  • the gas exchange surface has a gas permeability of at least 10 cm 3 /m 2 .d.bar. This can enhance the mixing and oxygen transfer.
  • the nutrient supply unit has an aspect ratio of at least five. This can enhance the mixing and oxygen transfer.
  • the media distribution device and the media collection device are arranged to create a flow of media to maximizing contact of the culture media with the gas exchange surface. This can enhance the mixing and oxygen transfer.
  • the media distribution device is configured to induce a flow rate of the media entering the vessel to be of at least 1 % of the vessel working volume per hour. This can enhance the mixing and oxygen transfer.
  • the nutrient supply unit further comprises sensors for measuring media quality, oxygen concentration, or both, and for regulating oxygen diffusion accordingly. This can enhance the mixing and oxygen transfer.
  • the nutrient supply unit further comprises anchors for maintaining it in a vertical position when immersed in media. This can be particularly relevant when considering the combination of enhancing the mixing and oxygen transfer and enabling easier maintenance.
  • a bioreactor comprising a vessel and at least one nutrient supply unit according to the invention protruding in the vessel of the bioreactor.
  • bioreactor according to the invention it can optionally include one or more of the following characteristics alone or in combination:
  • the bioreactor comprises at least two nutrient supply units.
  • the value of the ratio of cumulated surface area of the gas exchange surface within a vessel on the volume of the vessel is of at least 50 cm -1 .
  • the invention relates to a cell culture system comprising at least one nutrient supply unit according to the invention or at least one bioreactor according to the invention.
  • the invention relates to a method for cell culture, using a nutrient supply unit comprising a gas exchange device, the gas exchange device comprising a gas exchange surface; a media distribution device; and a media collection device; preferably said nutrient supply unit protruding, at least partially in a vessel of a bioreactor; said method comprising: a step of introducing a gas into the vessel through the gas exchange device; a step of introducing a culture media into the vessel through the media distribution device; and a step of removing the culture media from the vessel through the media collection device.
  • the steps of introducing a culture media and removing the culture media induce a flow of culture media at the gas exchange surface.
  • the steps of introducing a culture media and removing the culture media can be done simultaneously.
  • This method allows a gentle yet effective mixing, ensuring cell viability and integrity.
  • the present method can be used to reduce production costs particularly in applications requiring large volumes of culture media, such as the production of cultivated meat or large-scale vaccine manufacturing.
  • the invention relates to a cell biomass obtainable from a method according to the invention, said cell biomass comprising less than 50 ppm of shear-stress protectant agent.
  • the invention relates to an edible food product obtainable from cell biomass according to the invention, said edible food product comprising less than 45 ppm of shear-stress protectant agent.
  • Figure 1 is a schematic view of a nutrient supply unit according to an embodiment of the invention.
  • Figure 4 is a schematic view of a cell culture system according to an embodiment of the invention.
  • the functions associated with the box may appear in a different order than indicated in the drawings.
  • two boxes successively shown may be performed substantially simultaneously, or boxes may sometimes be performed in the reverse order, depending on the functionality involved.
  • Cultivated cells can originate from cells of any origin such as cells from biopsies, from stem cells isolated from animal embryos, or correspond to stem cells themselves. Cells can be cultivated as single cells, cell clusters, organoids, spheroids, or on microcarriers.
  • the terms “media” or “medium” are used interchangeably. They can refer, within the meaning of the invention, to a liquid, a growth medium, a culture medium, or an environment allowing the growth, proliferation, differentiation and maintenance of microorganisms and/or cells.
  • the media can include nutrients.
  • the media can also include substance, small molecule or compound inducing or controlling the differentiation of the cultivated cells.
  • nutrient can refer, within the meaning of the invention, to any substance, small molecule or compound that provides nourishment essential for the maintenance of life and/or for growth.
  • the term nutrient broadly comprises both macronutrients and micronutrients. These encompass essential nutrients like amino acids, vitamins, minerals, proteins, carbohydrates, fats and/or oxygen.
  • the term “vessel” can refer, within the meaning of the invention, to a container or chamber designed to host and maintain biological reactions, particularly for the cultivation of animal cells.
  • This vessel is typically part of a larger bioreactor system and is engineered to provide an optimal environment for cell growth, eventually differentiation and proliferation within a cultivation volume.
  • the vessel working volume (or cultivation liquid volume) can refer to the vessel internal volume subtracted by the nutrient unit(s) volume and the eventual gas headspace. It generally corresponds to the volume of liquid inside the working vessel.
  • the vessel cell bed volume can refer, when considering fluidized bed reactors, to the volume occupied by the cells during growth conditions. It should be about 90% of the liquid volume.
  • gas exchange can refer, within the meaning of the invention, to the process by which gases are transferred for example across an element.
  • This gas exchange element comprising generally a gas exchange surface, which can equilibrate the concentration of certain gaseous molecules between two phases.
  • Gas exchange for example across membranes, typically occurs via diffusion. This means that gases move from an area of higher concentration to an area of lower concentration. The rate of diffusion usually depends on the concentration gradient across the element (e.g. membrane), the permeability of the membrane to each gas, and the physical properties of the gases themselves.
  • backbone structure can refer, within the meaning of the invention to a component, a rigid central framework within a gas exchange device, designed to provide essential structural support and stability. It can be characterized by its sturdy construction and is often the primary load-bearing element. This structure not only supports the physical components of the gas exchange device but also houses and protects critical functional elements like distribution channels or gaseous exchange membrane. Despite its rigidity, the structure is hollow, allowing for efficient passage and distribution of substances necessary for the bioreactor's operation.
  • biological products can refer, within the meaning of the invention, to any products derived from living organisms or cells, encompassing cells themselves, cell biomass, and cell products.
  • biologies can be composed of sugars, proteins, or nucleic acids or complex combinations of these substances, or may be living entities such as cells or tissues.
  • the large-scale cell production such as animal cells for cultivated meat, cultivated leather or large-scale vaccine manufacturing requires enhanced mixing and oxygen transfer, reduced shear stress and scalability for industrial production.
  • the inventors have created a nutrient supply unit designed to efficiently distribute the nutrient and, in particular, oxygenate the media at a reduced cost.
  • This innovation especially suited for fluidized bed bioreactors, paves the way for large-scale cell production that is economically feasible.
  • the invention relates to a nutrient supply unit 1 for bioreactor 100.
  • the nutrient supply unit 1 according to the invention is preferably adapted for use in a bioreactor for in suspension culture.
  • the nutrient supply unit 1 according to the invention is preferably adapted for use as a protruding entity, preferably at least partially, in a vessel of a bioreactor.
  • a nutrient supply unit 1 for bioreactor 100 may comprise a gas exchange device 10, a media distribution device 20 and a media collection device 30.
  • the introduction of media into the vessel 110 can comprise the use of pulses for example, at the onset of the operation. These pulses can vary in duration and intensity. This initial pulsing action is designed to rapidly equilibrate the vessel 110 with the necessary nutrients and conditions required for cell culture. The pulsing nature of the flow ensures that the media is mixed thoroughly, preventing gradients of nutrients, gases, or waste products from developing. This is particularly important in the early stages of cell culture, where cells are adapting to their environment and require a homogenous mixture for optimal growth. However, in standard operational regime, media can be introduced into the cell culture vessel at a constant rate. This ensures a steady supply of nutrients and efficient removal of waste products, creating a stable environment conducive to sustained cell growth.
  • the nutrient supply unit 1 may comprise a hollow structure, preferably a hollow cylindrical structure.
  • a hollow structure can refer to a three-dimensional form with at least one internal cavity.
  • a nutrient supply unit 1 may comprise at least two internal cavities.
  • the nutrient supply unit 1 can comprise a structure, such as hollow structure formed by additive manufacturing. Indeed, when the hollow structure corresponds at least to the gas exchange device 10, media distribution device 20 and media collection device 30, it can be advantageously manufactured by additive manufacturing.
  • the nutrient supply unit 1 may have an aspect ratio of at least two.
  • An aspect ratio is defined as the ratio between the external length of the nutrient supply unit 1 and its external diameter.
  • the nutrient supply unit 1 may have an aspect ratio of at least five, preferably at least seven and more preferably an aspect ratio of at least ten.
  • the gas exchange surface 12 can be directly formed by the walls of the internal cavity 11.
  • the cavity 11 may include openings leading to a gas exchange membrane.
  • the gas exchange device 10 comprises a gas exchange surface 12 that comprises or consists in a gas exchange membrane such as a hydrophobic gas-permeable wall.
  • the monitoring and adjustment system 500 can comprise sensors and control devices distributed throughout the cell culture system 2 to continuously monitor environmental parameters like pH, temperature, nutrient concentration including oxygen levels, pressure and waste or waste metabolites level.
  • the collected data can be used to adjust conditions in real-time, ensuring uniformity in nutrient distribution for example.
  • the monitoring and adjustment system 500 can be configured to control the pressure of the gaseous phase providing the oxygen supply.
  • the transport of oxygen at the gas exchange surface can scale with the external gas pressure.
  • a system 2 according to the invention can comprise centrifugal separators 720. These devices use centrifugal force to separate cells from the culture medium. The centrifugal force causes cells to aggregate and separate from the less dense medium. This method is particularly efficient for large-scale production.
  • centrifugal separators 720 can be used to isolate harvested cells from culture medium. The centrifugal separators 720 operate on the principle of centrifugal force to effectuate the separation of cells from the culture medium. When the culture medium is subjected to this force, cells, being denser, aggregate and separate from the less dense medium.
  • a system 2 can comprise a harvesting system 800.
  • the harvesting system 800 is adapted for collecting biological products from large volumes without compromising quality and eventually asepsis.
  • the harvesting system 800 can include separation technologies, like membrane filtration or centrifugation, integrated within the cell culture system 2.
  • the outflow volumetric flow rate has a maximum value equal to the maximum specific growth rate characterized by the type of cell, multiplied by the total reactor volume.
  • the cell culture within the bioreactor 100 is closely monitored to determine the optimal time for biological products collection.
  • the biological products are harvested when the cell culture reaches a desired density and viability status.
  • the harvesting can be done while the bioreactor 100 is operating or after that the fluidization has been paused.
  • the cessation of the fluidizing elements allows for the cells to transition from a growth phase to a state that is more conducive to harvesting.
  • the culture media comprising the cell biomass can be drained from the bottom of the vessel 110.
  • biological products can be subjected to a centrifugation process. This step is designed to separate the cells from any remaining culture medium and to concentrate the cell biomass.
  • the resulting cell pellet can then be subjected to a washing process using for example a sterile buffer solution. The purpose of this washing process is to remove any impurities or residual media components, further purifying the cell biomass.
  • a method 1000 for cell culture comprises the use of at least one nutrient supply unit 1 according to the invention or at least one bioreactor 100 according to the invention or at least one cell culture system 2 according to the invention.
  • a method 1000 for cell culture can comprise the use of a nutrient supply unit 1 comprising a gas exchange device 10, the gas exchange device 10 comprising a gas exchange membrane 12; a media distribution device 20; and a media collection device 30; said nutrient supply unit 1 protruding in the vessel 110 of the bioreactor 100.
  • a method according to the invention preferably comprises a step of introducing a gas 1100 into the vessel 110, a step of introducing a culture media 1200 into the vessel 110, and a step of removing the culture media 1300 from the vessel; said steps of introducing a culture media 1200 and removing a culture media 1300 being simultaneous and inducing a flow of culture media at the gas exchange membrane 12 surface.
  • the present invention can also relate to a biological product obtainable from a method according to the invention.
  • the present invention can also relate to a cell biomass obtainable from a method according to the invention. More preferably, the present invention can relate to a cell biomass obtained from a method according to the invention.
  • the shear-stress protectant agent is a polyoxyethylenepolyoxypropylene block copolymer such as a poloxamers.
  • the shear-stress protectant agent is a triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).
  • these compounds are known anti-foaming agents that can be used in cell cultivation.
  • the shear-stress protectant agent is a poloxamer (also called Pluronic®), such as Poloxamer 188.
  • the present invention can also relate to an edible food product obtainable from cell biomass according to the invention.
  • the invention relates to an edible food product obtained from, and/or comprising, cell biomass according to the invention.

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  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne une unité d'alimentation en substances nutritives (1) pour un bioréacteur (100) comprenant une cuve (110), ladite unité d'alimentation en substances nutritives (1) comprenant : un dispositif d'échange de gaz (10), le dispositif d'échange de gaz (10) présentant une surface d'échange de gaz (12) et étant conçu pour introduire un gaz dans le bioréacteur (100), de préférence sans induire de contrainte de cisaillement dans le bioréacteur (100) ; un dispositif de distribution de milieu (20), le dispositif de distribution de milieu (20) étant conçu pour introduire un milieu de culture dans le bioréacteur (100) ; et un dispositif de collecte de milieu (30), le dispositif de collecte de milieu (30) étant conçu pour faire sortir le milieu de culture du bioréacteur (100) ; ledit dispositif d'échange de gaz (10), le dispositif de distribution de milieu (20) et le dispositif de collecte de milieu (30) étant agencés pour induire un écoulement de milieu de culture au niveau de la surface d'échange de gaz (12).
PCT/EP2024/088600 2023-12-28 2024-12-27 Unité de distribution de substances nitritives pour bioréacteur Pending WO2025141180A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23307418 2023-12-28
EP23307418.6 2023-12-28

Publications (1)

Publication Number Publication Date
WO2025141180A1 true WO2025141180A1 (fr) 2025-07-03

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63240774A (ja) * 1987-03-27 1988-10-06 Ube Ind Ltd バイオリアクタ−
US5149649A (en) * 1989-08-04 1992-09-22 Mitsubishi Rayon Co., Ltd. Multi-layered porous hollow fiber membrane for use in cell culture
US5601757A (en) * 1994-08-24 1997-02-11 Forschungszentrum Julich Gmbh Gasification-tube module and reactor for cell cultivation
WO2023156933A1 (fr) * 2022-02-16 2023-08-24 Future Meat Technologies Ltd. Systèmes et procédés pour le régénération d'un milieu de culture cellulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63240774A (ja) * 1987-03-27 1988-10-06 Ube Ind Ltd バイオリアクタ−
US5149649A (en) * 1989-08-04 1992-09-22 Mitsubishi Rayon Co., Ltd. Multi-layered porous hollow fiber membrane for use in cell culture
US5601757A (en) * 1994-08-24 1997-02-11 Forschungszentrum Julich Gmbh Gasification-tube module and reactor for cell cultivation
WO2023156933A1 (fr) * 2022-02-16 2023-08-24 Future Meat Technologies Ltd. Systèmes et procédés pour le régénération d'un milieu de culture cellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CELIK, YUNUS ET AL.: "A novel low shear horizontal bioreactor design for the production of animal cells: Effect of bioreactor dynamics on the 3D spheroid formation of HepG2.", BIOCHEMICAL ENGINEERING JOURNAL, vol. 196, July 2023 (2023-07-01), pages 108952
JARA, T.C.PARK, K.VAHMANI, P. ET AL.: "Stem cell-based strategies and challenges for production of cultivated meat.", NATURE FOOD, vol. 4, October 2023 (2023-10-01), pages 841 - 853
ZHOU, TIANXUN ET AL.: "A review of algorithmic approaches for cell culture media optimization.", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, vol. 11, 11 May 2023 (2023-05-11), pages 1195294

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