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
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/14—Bioreactors or fermenters specially adapted for specific uses for producing enzymes
• • 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
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/44—Multiple separable units; Modules
• • 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
  • C12M29/10—Perfusion
• • 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
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/10—Separation or concentration of fermentation products
• • 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
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/12—Purification

Definitions

• the present invention relates to the field of biopharmaceutical manufacturing, in particular that of protein production. More specifically, the present invention relates to a modular cell-based production system designed for the production of monoclonal antibodies and other therapeutic proteins. The invention further relates to a perfusion bioreactor, in particular a fully continuous and integrated bioprocess designed for the production of monoclonal antibodies and other therapeutic proteins.
• Therapeutic proteins including monoclonal antibodies (also referred to as “mAbs”) have been produced, at large-scale, for therapeutic, investigative and diagnostic purposes since the 1980s. They are produced using biological manufacturing processes (also referred to as a “bioprocess”) involving a number of unit operations starting with the growth of a cell line, (a section of the process traditionally referred to as “Upstream Processing” or “USP”) followed by the separation of the solids from the liquid supernatant onto which they are secreted (a section of the process traditionally referred to as “Primary Recovery”) up to the final purification steps (a section of the process traditionally referred to as “Downstream Processing”).
• Fig 1A shows a generic bioprocess used for the production of a biological product.
• the cells are grown at the laboratory scale (inoculum stage) and then used to seed a production bioreactor. Once the required cell growth and productivity have been achieved, the product is harvested using clarification processes such as centrifugation and/or filtration.
• the downstream processes traditionally begin with a capture chromatography step using protein A, followed by viral inactivation steps at low pH, followed by a neutralisation step using base.
• the product is then further purified using a series of ion exchange chromatography steps to remove impurities and contaminations. This is followed by a nanofiltration step to remove remaining viruses and viral particles, then by ultrafiltration and diafiltration steps to concentrate the protein of interest and placed in a suitable medium prior to bulk filling.
• the bulk filled product is then traditionally shipped to another site for filling in suitable items to make it ready for use by patients (vials, syringes, etc).
• Single-use equipment have the advantage of reducing capital expenditures (CAPEX), eliminating the needs for expensive clean-in-place (CIP) and sterilise-in-place (SIP) processes, as well as eliminating certain required equipment (CIP skids, clean steam generators, increase waste holds, etc.) which will reduce the footprint of the production plant.
• CIP clean-in-place
• SIP sterilise-in-place
• the present invention provides a modular cell-based production system in the form of a physical “skid” or “unit” providing a fully continuous and integrated bioprocess designed for the production of monoclonal antibodies and other therapeutic proteins.
• This system is implemented as a physical unit, or a modular “skid”, which can host separate aspects of the bioprocesses, mainly Bioprocess 1 : the cell culture, primary recovery and a chromatography capture step (such as protein A affinity chromatography); and Bioprocess 2: the viral inactivation and subsequent a chromatography step (such as ion exchange chromatography) (see Fig. 1B and C).
• a “skid” comprises a first module and a second module which are in fluid contact with each other.
• a “skid” can be used for either bioprocess 1 (combination of USP and DSP) or bioprocess 2 (DSP) and this enables to combine both upstream and downstream processes. Since the skid is designed multi-purpose, the first module can be used as a bioreactor in bioprocess 1 or can be used for the viral inactivation step in bioprocess 2. The second module can be used for a chromatography step such as protein A chromatography in bioprocess 1 or for example ion exchange chromatography in bioprocess 2.
• the skid is designed to be multi-purpose, so as to accommodate either of the 2 aforementioned process segments or others. Moreover, the system is designed to host an integrated and fully continuous bioprocess. This is a novelty in the field as (1 ) there are currently no fully continuous processes implemented in biopharmaceutical manufacturing; (2) there are currently no offerings for an integrated continuous bioprocess system combining both upstream and downstream processes; (3) there are currently no skids that offer the modularity and flexibility to combine different unit operations or process equipment.
• the present invention has a single-use flow path, meaning that all parts which come into contact with product fluid or exposed to it, can be/are single-use / disposable made of medical grade component plastic or polymer (such as polyethylene, polycarbonate, polypropylene, polyamide, silicone, etc. and are a consumable.
• the present invention provides a modular cell-based protein production system comprising:
• a first module designed to perform one or more bioprocesses selected from the list comprising: cell culturing and viral inactivation;
• chromatography designed to perform one or more bioprocesses selected from the list comprising: chromatography; in particular protein A chromatography, ion exchange chromatography, size- exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two- dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular protein A chromatography or ion exchange chromatography; wherein in operation of said system, said first and second module are in fluid contact with each other.
• chromatography in particular protein A chromatography, ion exchange chromatography, size- exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two- dimensional chromatography
• the present invention provides a modular cell-based protein production system comprising:
• a first module designed to perform one or more processes selected from the list comprising: cell culturing, viral inactivation and bioconjugation;
• bioprocess 1 comprising cell culturing in the first module and chromatography in the second module
• bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.
• said first module is designed to perform all of the processes from the list comprising: cell culturing and viral inactivation; and said second module is designed to perform at least all of the processes from the list comprising: protein A chromatography, and ion exchange chromatography.
• said first module comprises:
• said second module comprises:
• said system by means of the combination of said first and second module, allows to perform a first bioprocess comprising cell culturing in the first module and chromatography in the second module.
• said container of the first module of the first bioprocess accommodates a cell culture suspension or adherent cells.
• said stirring means of the first module of the first bioprocess is in the form of a cylindrical rotating filter.
• said one or more ports of the first module of the first bioprocess are selected from the list comprising: a) a port for introduction of a cell culture medium; b) a port for the introduction of a cell culture suspension; c) a port for the removal of excess cell culture suspension; d) a port for the removal of clarified harvest; e) a port for sample removal of cell culture suspension; f) a port for the sterile introduction of gases; g) a port for gas exhaust; h) ports for sensors and analytical purposes.
• said chromatography columns of the second module of the first bioprocess allow for chromatography in particular protein A chromatography, ion exchange chromatography, size-exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular protein A chromatography capturing.
• said system by means of the combination of said first and second module, allows to perform a second bioprocess comprising viral inactivation in the first module and chromatography in the second module.
• said container of the first module of the second bioprocess accommodates a process fluid containing the protein.
• said stirring means of the first module of the second bioprocess is in the form of a magnetic agitator.
• said one or more ports of the first module of the second bioprocess are selected from the list comprising: a) a port for the introduction of a process fluid containing the protein; b) a port for the introduction of a base; c) a port for the introduction of an acid; d) a port for the removal of inactivated process fluid; e) a port for the introduction of a detergent; f) a port for sample removal; g) a port for the sterile introduction of gases; h) a port for gas exhaust; i) ports for sensors and analytical purposes.
• said chromatography columns of the second module of the second bioprocess allow for chromatography in particular protein A chromatography, ion exchange chromatography, size-exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular ion exchange chromatography.
• the present invention provides a perfusion bioreactor suitable for use as a first module in the system as defined herein.
• the present invention provides a perfusion bioreactor comprising:
• Fig. 1 Schematic block flow diagram of a bioprocess for the manufacture of monoclonal antibodies (mAbs).
• Panel A represent the traditional prior art bioprocess subdivided in an upstream process (USP) and a downstream process (DSP).
• Panel B displays the bioprocess according to the present invention (referred to as bioprocess 1 and bioprocess 2).
• Panel C displays the interplay of the modules within the skid, and their relationship to the different aspects of the bioprocesses
• inoc inoculum stage
• VIN viral inactivation
• Neutralisation step
• IEX Chrom ion exchange chromatography
• NF nanofiltration
• UF ultrafiltration
• DF diafiltration.
• Fig.2 Schematic representation of the “skid” or “unit”.
• the physical implementation of the invention is a “skid” or a “unit”, with 2 main “segments” or “modules” occupying a physical space of the skid each;
• First module or “Segment A” hosting the bioreactor in bioprocess 1 or hosting the viral inactivation step in bioprocess 2.
• Second module or “Segment B” hosting the chromatography step such as protein A chromatography in bioprocess 1 or hosting the chromatography steps such as ion exchange chromatography in bioprocess 2.
• Bioprocess 1 is particularly characterized by the use of the first module as a perfusion bioreactor, and the use of the second module in as chromatography capturing step e.g. protein A capture step.
• Bioprocess 2 is particularly characterized by the use of the first module in the viral inactivation step and the use of the second module in chromatography steps such as the ion exchange chromatography.
• Fig. 4 Schematic of the perfusion bioreactor.
• the perfusion bioreactor of the present invention in particular comprises: a container for accommodating a fluid; a stirring means for stirring said fluid; one or more capillary fibers or membranes for separating cells from the surrounding liquid medium; and one or more ports for introducing or removing components in/from said container; wherein said container is in particular designed as a consumable made from a single-use material, such as plastic.
• DO dissolved oxygen
• IR infrared spectroscopy.
• the present invention provides a modular cell-based protein production system comprising:
• a first module designed to perform one or more bioprocesses selected from the list comprising: cell culturing and viral inactivation;
• chromatography designed to perform one or more bioprocesses selected from the list comprising: chromatography; in particular protein A chromatography, ion exchange chromatography, size- exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two- dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular protein A chromatography or ion exchange chromatography; wherein in operation of said system, said first and second module are in fluid contact with each other.
• chromatography in particular protein A chromatography, ion exchange chromatography, size- exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two- dimensional chromatography
• the present invention provides a modular cell-based protein production system comprising:
• a first module designed to perform one or more processes selected from the list comprising: cell culturing, viral inactivation and bioconjugation;
• bioprocess 1 comprising cell culturing in the first module and chromatography in the second module
• bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.
• the present invention provides a modular cell-based protein production system comprising:
• a first module designed to perform one or more processes selected from the list comprising: cell culturing, and viral inactivation;
• bioprocess 1 comprising cell culturing in the first module and chromatography in the second module
• bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.
• said first module is designed to perform all of the processes from the list comprising: cell culturing, bioconjugation and viral inactivation; and said second module is designed to perform at least all of the processes from the list comprising: protein A chromatography and ion exchange chromatography.
• said first module is designed to perform all of the processes from the list comprising: cell culturing, and viral inactivation; and said second module is designed to perform at least all of the processes from the list comprising: protein A chromatography and ion exchange chromatography.
• bioprocess is meant to be a method to manufacture a biological molecule using biological processes (such as fermentation, cell culture) in combination with other traditional methods within the chemical or industrial sectors, such as chromatography, filtration, etc.
• biological processes such as fermentation, cell culture
• other traditional methods within the chemical or industrial sectors such as chromatography, filtration, etc.
• manufacturing monoclonal antibodies and other therapeutic proteins involves an upstream process (inoculation of cells, cell culturing in a bioreactor, harvesting using clarification processes) and a downstream process (e.g. initial purification by protein A affinity chromatography, viral inactivation, neutralisation, purification by e.g. ion exchange chromatography steps, nanofiltration, ultrafiltration and diafiltration, and bulk filling) (Fig. 1 panel A).
• the term “protein” is meant to be a large biomolecule or macromolecule that is composed of one or more chains of amino acid residues. A single linear chain of amino acid residues is called a polypeptide, and accordingly a protein contains at least one polypeptide chain.
• a protein can be naturally produced by the cell or, can be a “recombinant protein”, produced by introducing the genetic code of the protein via plasmid DNA into a host cell. This host cell can be a CHO, HEK, NS0, hybridoma, Sf9 or any other animal cell line.
• the term “monoclonal antibody” or “mAb” is meant to be a protein used for therapeutic or diagnostic purposes. It can be an antibody that was traditionally made by cloning of unique white blood cells, but is currently mostly made using recombinant methods.
• the term “culturing” is meant to be the process in which cells are grown and/or allowed to multiply. Said culturing can be done as a free-floating cell culture, meaning that the cells are not attached to a support. Alternatively the cells may be attached to support.
• the perfusion bioreactor as used herein comprises a spin filter, suitable for separating cells from the surrounding liquid medium.
• the term “viral inactivation” is meant to be a step of the claimed protein production methods in which virus contaminants are inactivated. This can be achieved by subjecting the bioprocess fluid to conditions that denature the virus protein but not the active ingredient.
• this step can suitably be performed in the first module (segment A) of the claimed device, which comprises a stirring means.
• bioconjugation is meant to be a chemical strategy to form a stable covalent link between two molecules, at least one of which is a biomolecule.
• Bioconjugation chemistry relies on a series of well-controlled steps in sequence which may include functional group reduction, activation, API-linker conjugation to the primary biologic and any number of wash or solvent or buffer exchange steps throughout.
• Bioconjugate molecules are a class or generation of biologic molecules which are designed to have an increased efficacy enabled by the combined function of two or more different therapeutic types of molecules.
• Antibody-Drug Conjugates are one of the more common bioconjugates and are synthesized by biochemically modifying an antibody and covalently linking it to another active pharmaceutical ingredient (API).
• NF nanofiltration
• u I traf i I tratio n/d iaf i Itratio n is meant to be a combination of process steps where the product liquid is concentrated by membranes and the liquid replaced with a new solution.
• downstream processing or “DSP” is meant to be a series of process steps traditionally comprising the second half of the manufacturing process (purification steps).
• upstream processing or “USP” is meant to be a series of process steps traditionally comprising the first half of the manufacturing process (cell culture and cell harvesting steps).
• bioprocess 1 refers to the steps: cell culturing in a bioreactor, harvesting using clarification processes, and chromatography, in particular protein A chromatography, ion exchange chromatography, size-exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular protein A affinity chromatography (Fig. 1 Panel B).
• bioprocess 2 refers to the steps: viral inactivation, neutralisation, purification by chromatography, in particular protein A chromatography, ion exchange chromatography, size-exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, or aqueous normal-phase chromatography; more in particular ion exchange chromatography (Fig. 1 Panel B).
• Bioprocess 1 and bioprocess 2 can be implemented in the modular cell-based protein production system of the present invention.
• system or “skid” or “unit” is meant to be a platform providing a fully continuous and integrated bioprocess designed for the production of monoclonal antibodies and other therapeutic proteins.
• it is an integrated physical unit where the main equipment necessary for the manufacturing of the product (such as bioreactors, chromatography columns, etc.) is located, as well the secondary / auxiliary equipment (such as pumps, pinch valves, transmitters, etc.) (Fig. 2).
• the physical implementation of the invention is a “system” or “skid” or a “unit”, with 2 main “segments” or “modules” occupying a physical space of the skid each.
• first module is meant to be segment A which may host one or more containers such as bioreactor(s) when used in connection with bioprocess 1 (Fig. 3 panel A, left element) or may host one or more containers such as viral inactivation tankswhen used in connection with bioprocess 2 (Fig. 3 panel B, left element).
• second module is meant to be segment B which may host one or more chromatography columns and/or membranes for e.g. the protein A chromatography when used in connection with bioprocess 1 (Fig. 3 panel A, right element) or may host one or more chromatography columns and/or membranes for e.g. the ion exchange chromatography steps when used in connection with bioprocess 2(Fig. 3 panel B, right element).
• the skid/unit may have HEPA (or alternative) filters installed above, for purifying ambient air to provide a local clean environment, at least equal to the environment obtained in a pharmaceutical cleanroom of any of the following grades: class D, class C, class B and/or class A.
• said system by means of the combination of said first and second module, allows to perform a first bioprocess comprising cell culturing in the first module and chromatography in the second module.
• said system by means of the combination of said first and second module, allows to perform a second bioprocess comprising viral inactivation in the first module and chromatography in the second module.
• module is meant to be a modular design wherein each component can be removed and replaced with a similar component.
• a central aspect of the skid design is its modularity and interchangeability meaning that the different segments can accommodate different processes with minimal modifications from the end-user, not requiring interventions from the vendor or supplier, such as: (i) installing the correct components (consumables) and making the connections (ii) installing adaptors or spool pieces, (iii) modifying the control software parameters. All equipment are consolidated and integrated in one physical unit, controlled under one automation system (a single Programmable Logic Controller)
• said first module of the first bioprocess comprises:
• fluid is meant to be substance that continually deforms (flows) under applied shear stress, or external forces. Fluids are a phase of matter including liquids, gases, plasmas,..., however in the context of the present invention the term ‘fluid’ is in particular used in connection with a liquid.
• the term “container” refers to “bioreactor” when used in the context of the first module of bioprocess 1 ; while the term “container” refers to “viral inactivation tank” when used in the context of the first module of bioprocess 2.
• the term “bioreactor” is meant to be a container and integral vessel suitable for growing cells or micro-organisms.
• the cell culture bioreactor may be a gamma irradiatable/sterilisable single-use / disposable chamber made of a rigid plastic or polymer such as polyethylene, polycarbonate, polypropylene, polyamide, polysiloxanes, polystyrene, polyvinyl chloride, synthetic rubber, phenol formaldehyde resin (or Bakelite), neoprene, nylon, polyacrylonitrile, polyvinylbutyral.
• the bioreactor process is designed for the culturing of cells in suspension (e.g.
• the separated cell-free liquid is harvested through a spin filter-type device or through any of the established cell separation perfusion methods (such as tangential flow filtration, flocculation, etc) or other methods.
• This chamber could either be cylindrical, with baffles to aid the mixing, or be of a rectangular or square shape.
• the chamber will have a working volume of at least about 1 L, such as 10 L, 20L, 30L, 40L, 50L, 100L, 200L, 300L, 400L, 500L, 600L, 700L, 800L, 900L, 1000L.
• said container of the first module of the first bioprocess or “bioreactor” accommodates a cell culture suspension or adherent cells.
• viral inactivation tank is meant to be a container and integral vessel suitable for viral inactivation.
• the viral inactivation tank may be installed in the first module (segment A) of the system onto a magnetic device such as a magnetic agitator. It may use the magnetic drive unit also used by the bioreactor to drive agitation/mixing during the inactivation process.
• This container may be removable.
• the virus-inactivation process usually takes place in a stirred tank in which pH, hold time, and temperature are controlled. After adjusting pH to the desired acidic value (about and between 3 to 5), operators incubate the tank contents for a specified duration at a specified temperature to achieve effective virus inactivation. Afterward, the solution is neutralized using a base to a pH about and between 5 to 8, or as required for the next processing step.
• said container of the first module of the second bioprocess or “viral inactivation tank” accommodates a process fluid containing the protein.
• process fluid is meant to be liquid containing the product of interest, which is a protein of therapeutic or diagnostic value.
• a “stirring means” is referred to as any rotating device acting as a mechanical mixing device to ensure a homogenous mixture.
• said stirring means may be in the form of a spin filter such as a rotating cylindrical filtration device made of a gamma irradiatable/sterilisable sintered porous rigid medical grade element (plastic or other), with a porosity of 1 - 100 pm.
• the “spin filter” serves at least 2 purposes i.e.
• the spent medium fluid containing the product of interest (referred to as the “clarified harvest”) from the cell suspensions or microcarriers onto which cells are adhered; and acting as a mechanical mixing device to ensure that the bioreactor contents are homogeneous, helping with oxygen transfer and an efficient temperature distribution.
• the material of the spin filter membrane is selected from the list comprising: microporous silica, polyether sulfone (PES), polytetrafluoroethylene (PTFE/Teflon), polyvinilydene fluoride (PVDF), polyethylene or nylon/polyamide.
• said spin filter membrane may also comprise a hollow, cylindrical fiber material.
• the spin filter may be manufactured using 3D printing methods. In opposition to other bioreactor systems, the spin filter is not meant to provide a growth surface for adherent cells. As cell density increases, a film of cells will form on the external surface of the spin filter.
• said stirring means of the first module of the first bioprocess is in the form of a cylindrical rotating filter.
• said stirring means of the first module of the second bioprocess may be in the form of a magnetic device such as a magnetic agitator, magnetic stirrer or in the form of an overhead stirrer.
• a number of parameters can be monitored on-line (i.e. in the bioreactor) and/or controlled, which could include but are not limited to the following: pH, DO (dissolved oxygen), Temperature, Raman, IR (infrared spectroscopy), etc.
• Other parameters measured on-line or at-line (between the bioreactor and the chromatography step) include, but are not limited to, the titres of the protein of interest (the product, mAb).
• the bioreactor is equipped with a sparger for aeration purposes.
• a number of gases such as compressed air, oxygen, CO2 and N2 can be supplied to the bioreactor.
• the spin filter can be fixed on the bottom of the bioreactor via a ball bearing mechanism allowing it to rotate securely. Agitation may be magnetically driven.
• the ball bearing mechanism may comprise 2 races: an external rotating one comprising the spin filter, and an internal stationary one fixed onto the bioreactor chamber.
• the balls may be of spherical shape, with a diameter of about 1-2 mm made of an inert material such as ceramic. They may be located and roll in a groove between the outer compartment (spin filter) and the inner compartment.
• the ball bearing mechanism is designed to provide a sealed barrier to prevent cells from going into the clarified harvest chamber (inside the spin filter). However, should this occur, the quantity can be further reduced via a number of mechanisms, such as for example: (i) a built-in series of channels to collect cells and direct them to the outside of the spin filter.
• the rotation of the spin filter can be magnetically driven.
• An external drive-unit comprising the magnetic plate onto which the bioreactor sits and drives the magnetic agitation.
• the bioreactor may be placed on a weighing platform to measure the weight (and by extrapolation the volume) of the contents.
• the system may have a location to store fresh media in bags, to be fed into the bioreactor.
• the system may have a location to contain a bag to store biological waste.
• the different modules of the present invention may contain one or more ports.
• said one or more ports of the first module of the first bioprocess are selected from the list comprising: a) a port for introduction of a cell culture medium; b) a port for the introduction of a cell culture suspension; c) a port for the removal of excess cell culture suspension; d) a port for the removal of clarified harvest; e) a port for sample removal of cell culture suspension; f) a port for the sterile introduction of gases; g) a port for gas exhaust; h) ports for sensors and analytical purposes.
• said one or more ports of the first module of the second bioprocess are selected from the list comprising: a) a port for the introduction of a process fluid containing the protein; b) a port for the introduction of a base; c) a port for the introduction of an acid; d) a port for the removal of inactivated process fluid; e) a port for the introduction of a detergent; f) a port for sample removal; g) a port for the sterile introduction of gases; h) a port for gas exhaust; i) ports for sensors and analytical purposes.
• the different ports for addition of withdrawal of components may be equipped with a mechanism for controlling the amount of material flowing through the different ports.
• pinch valves may be installed on tubings attached to the different ports. Pinch valves (non-product contact) can be used to close/open the flow path as required by pinching the tubing closed and open. The combination of tubing / manifold set-up and pinch will help to direct the fluid flow, such as in the continuous chromatography steps.
• a fluid flow can be made in any of the following methods, or combinations thereof:
• tubing / hoses made of silicone or thermoplastics
• a chromatography technique may be performed in the second module of the first bioprocess as well as the second bioprocess.
• said second module comprises:
• buffer solution is meant to be different salt-based solutions used in various steps of the purification processes, as liquid phases.
• said chromatography columns of the second module of the first bioprocess allow for chromatography such as protein A chromatography capturing.
• said chromatography columns of the second module of the second bioprocess allow for chromatography such as ion exchange chromatography.
• chromatography is meant to be a technique used for the separation of a mixture, wherein the mixture is called the mobile phase, which is carried through a system such as a column, capillary tube, plate, sheet,... termed the stationary phase.
• the molecules to be separated stay longer or shorter on the stationary phase, depending on their interaction therewith. Accordingly, these molecules travel at different velocities in the mobile phase, thereby causing them to separate.
• the chromatography techniques in the bioprocess 1 and 2 may be independently selected from the list comprising: protein A chromatography, ion exchange chromatography, size-exclusion chromatography, expanded bed adsorption chromatographic separation, reversed-phase chromatography, hydrophobic interaction chromatography, hydrodynamic chromatography, two-dimensional chromatography, simulated moving-bed chromatography, pyrolysis gas chromatography, fast protein liquid chromatography, countercurrent chromatography, periodic counter-current chromatography, chiral chromatography, aqueous normal-phase chromatography; in a particular embodiment, said chromatography techniques are selected from protein A chromatography or ion exchange chromatography.
• protein A chromatography is meant to be a chromatography techniques relying on the specific and reversible binding of antibodies to an immobilized protein A ligand.
• the DSP part may comprise an affinity chromatography step, which may comprise of a solid phase (resin or membrane) with a protein A ligand for the capture of the protein produced upstream.
• an affinity chromatography step which may comprise of a solid phase (resin or membrane) with a protein A ligand for the capture of the protein produced upstream.
• a chromatography process can be operated in one of 2 modes:
• Bind & Elute the product of interest (e.g. the mAb) is captured on the solid phase, while process fluids go to waste • Flow-through: this is the opposite, whereby the matrix captures some impurities and contaminants and the product of interest is not captured
• the protein A chromatography step may be performed in bind & elute mode
• the USP and DSP steps can be separated by a sterilizing filter (e.g. 0.22 pm porosity normally), which forms a “sterile barrier”
• a sterilizing filter e.g. 0.22 pm porosity normally
• ion exchange chromatography is meant to be a process which separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecules, such as large proteins, small nucleotides, amino acids, antibodies,... Ion exchange chromatography is typically done at conditions that are one unit away from the isoelectric point of the molecules.
• Ion exchange chromatography There are in principle 2 types of ion exchange chromatography, i.e. anion exchange, where the molecules are negatively charged and the stationary phase is positively charged; or cation exchange, where the molecules are positively charged and the stationary phase is negatively charged.
• the present invention provides a perfusion bioreactor suitable for use as the first module in the system as defined herein.
• the present invention provides a perfusion bioreactor comprising:
• Bioprocess 1 cell culturing and chromatography purification
• One or more bioreactor(s) may be installed in segment A of the system.
• the following parameters may be monitored in the bioreactor: pH, dissolved oxygen (DO), temperature, off-gas Metabolites), bioreactor contents (weight or volume), ....
• the following table shows a list of all parameters that may be monitored and/or controlled during the bioreactor step: Measuring of the parameters may be done via sensors that include but are not limited to the following: pH probe, DO probe, temperature probe, Raman spectroscopy, IR, biomass probe.
• the pH probe and DO probe may be either single-use (fluorescent patches or electrochemical) or reusable.
• the system may also be equipped with a biomass probe to measure the cell concentration. This could be done by measuring capacitance or other methods such as turbidity.
• the biomass probe may be single-use or re-usable.
• Temperature may be maintained in the bioreactor by an electrical heating element or a Peltier element positioned on the outside or inside of the bioreactor. Heating can potentially be performed via the drive unit plate located at the bottom of the bioreactor chamber.
• the system is set to maintain a temperature in the range of 20°C - 40°C.
• the volume inside the bioreactor may be kept constant by balancing the inflows (medium, buffer, etc.) with the outflows (clarified harvest, waste, etc.).
• the inflows rates can be set a constant speed by manual input, or automatically, feeding back from measured parameters such as biomass (measured via the biomass probe) or pH.
• the outflows rates can be set to equal the inflows or as a feedback from the contents (weight or volume) of the bioreactor.
• the volume of the contents of the bioreactor may be measured using weight (weighing platform on which sits the bioreactor)
• the bioreactor chamber is preferably a consumable, and may be sterilised externally by gamma irradiation, or alternative sterilisation means. It is taken out of its bag and installed onto the drive unit.
• a connection such as by flexible plastic tubing between the bioreactor chamber, and all relevant components such as fresh medium, harvest and waste may be used.
• the bioreactor chamber may be filled with medium, mixing is started, parameters control (temperature, pH, DO, etc) is started
• the bioreactor may be inoculated with a small flask (1 - 1000 ml_) of cell suspension (“the inoculum”) grown externally in an incubator
• clarified harvest can be removed from inside the spin filter for further processing, and fresh medium can be added at the same rate
• the perfusion rate which corresponds roughly to the number of medium exchanges per unit of time (e.g. volume / day) may be regulated by the flow rate of medium into the bioreactor and flow of clarified harvest out of the bioreactor plus the bleed rate (waste removal of cell suspension from outside the spin filter)
• Perfusion bioreactors have the advantage that they can culture cells over much longer periods of time compared to batch bioreactors. In particular, by continuously feeding the cells with fresh media and removing spent media while keeping cells in culture, the culture can be maintained for weeks or even months. In perfusion there are different ways to keep the cells in culture while removing spent media. One way is to keep the cells in the bioreactor by using capillary fibers or membranes, which the cells bind to.
• the purification segment of the process may comprise one or more of the following elements:
• a protein A chromatography step operated in bind & elute mode o this may comprise a number of columns (e.g. 3 to 5, such as 1 , 2, 3, 4 or 5) operated in continuous mode, using the existing simulated moving bed methodology
• the chromatography step may be set to primarily run in continuous mode but can also in non- continuous batch mode
• Continuous chromatography can be performed in 2 ways: o using the simulated moving method whereby the flow will be directed to multiple columns by means of valves control and flow path design o using a system whereby the columns are physically moved and connected/disconnected to/from the appropriate fluid path (process liquid, buffer, etc). This could be by means of a revolving mechanism involving a carousel or via other methods. In this set-up, there is a double shut-off mechanism to prevent leakages during column change. In the position ‘at rest’ , the fluid outlet towards the column (e.g. from the bioreactor) is closed and the fluid inlet to the column is also closed. Once the 2 parts are engaged, the fluid can flow across both segments.
• the fluid outlet towards the column e.g. from the bioreactor
• This mechanism is mainly made of solid plastic such as HDPE, and is aided by the use of springs and o-rings. This mechanism may use prior art, or existing systems.
• a number of buffer bags can be connected to the chromatography unit, these may comprise the following: wash buffer, elution buffer,
• the skid has a waste bag specific for downstream processing waste
• the chromatography columns mainly comprises acrylic glass but can also comprise other materials such as glass, borosilicate glass, or stainless steel.
• the columns may be pre-packed with the appropriate resin or comprise the appropriate membrane
• the clarified harvest coming out of the perfusion bioreactor and then into the sterile filter enters the first chromatography column from the top and is, in these steps referred to as the “feed”. This is the first processing step of the chromatography column and is referred to as the “loading of the column”.
• the product of interest will bind to the solid phase in the column. Once the first column is completely loaded (has reached its full binding capacity), feed flow is directed to the next column and so on. Any products that break through the first column may be directed into the next column, thus minimising product loss.
• the elution buffer is then pumped into the fully loaded column. Once the protein of interest is detected, the flow is directed down the process route to the next step, the viral inactivation unit.
• the protein A chromatography step operates using the simulated moving bed principle with the flow of the feed, the eluent, the product and the waste being directed across the different columns using the pinch valves mechanism
• bioprocess 2 of the present invention is further detailed using some specific examples.
• Viral Inactivation - Design One or more viral inactivation tank(s) can be installed in segment A of the system and may be as follows:
• a single-use plastic container rigid or soft, with a magnetic agitator onto which the product is transferred. It uses the magnetic drive unit also used by the bioreactor to drive agitation/mixing during the inactivation process.
• This container may be removable.
• the system uses the integrated pumps of the skid/unit to transfer the process fluid to the aforementioned container
• Viral inactivation is mainly done using pH adjustment.
• samples can be taken from the inactivation vessel, and pre-determined quantity of acid and base are then added.
• the weight of the vessel content may be monitored and the differential increase measured
• the viral inactivation, with pH adjustment, process may operate in a batch or semi-continuous step as follows:
• the process fluid is pumped into the viral inactivation tank. Acid is added in order to lower the pH to an appropriate value (around 3.5). In the meantime, the contents of the tanks are mixed by agitating the impeller.
• the purification segment of the process may comprise one or more of the following:
• An ion exchange chromatography step operated in bind & elute mode o this may comprise a number of columns (e.g. 3 to 5, such as 1 , 2, 3, 4 or 5) operated in continuous mode, using the existing simulated moving bed methodology or in multicolumn counter current solvent gradient purification (MCSGP) mode
• the chromatography step is set to primarily run in continuous mode but can also run in non- continuous batch mode
• a number of buffer bags can be connected to the chromatography unit, these comprise the following: wash buffer, elution buffer, equilibration buffer, acetate, phosphate, citrate, trifluoroacetic acid, tris (hydroxymethyl)- aminomethane, formic acid, ammonium formate, ammonium bicarbonate, and borate.
• the skid may have a waste bag specific for downstream processing waste • the chromatography columns, mainly comprise acrylic glass but can also comprise other materials such as glass, borosilicate glass, or stainless steel
• the elution buffer is then pumped into the fully loaded column. Once the protein of interest is detected the flow may be directed down the process route to the next step, such as the viral inactivation unit.
• the ion exchange chromatography step may operate using the simulated moving bed (SMB) principle with the flow of the feed, the eluent, the product and the waste being directed across the different columns using the pinch valves mechanism or by using the multicolumn counter current solvent gradient purification (MCSGP) mode.
• SMB simulated moving bed
• MCSGP multicolumn counter current solvent gradient purification

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• 2022
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