FIELD OF THE INVENTION

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.

BACKGROUND TO THE INVENTION

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”). This is illustrated in the following block flow diagram (Fig 1A.), showing 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).

Currently these therapeutic proteins are produced using batch processes, fed-batch processes or semi-continuous processes involving a perfusion upstream process followed by a number of batch subsequent steps. Single-use bioprocess equipment have been used in biopharmaceutical for a few decades, in the form of filters, then storage bags and then more recently since the beginning of the 2000s in the form of media and buffer mixers, bioreactors and downstream processing unit operations. Despite these developments, most mAbs are still produced using costly fixed stainless steel re-usable equipment, or a combination of stainless steel and single-use equipment. 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.

This combination of high CAPEX, high processing downtime and batch processing ultimately has an impact in the cost of goods sold (COGS) per unit (mg, g, kg, dose) of the therapeutic protein of interest and the cost incurred by the patient.

To overcome these drawbacks, the inventors of the present invention have developed an innovative platform for manufacturing biopharmaceuticals. In particular, 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).

As evident from Fig. 1C, 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.

Lastly, another advantage is the modular design such that each component can be removed and replaced with a similar component. 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,...) and are a consumable. SUMMARY OF THE INVENTION

In a first aspect, 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; and

- a second module 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.

In particular, 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; and

- a second module designed to perform chromatography; wherein in operation of said system, said first and second module are in fluid contact with each other thereby allowing to perform sequentially: a) bioprocess 1 comprising cell culturing in the first module and chromatography in the second module; and b) bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.

In a specific embodiment of the present invention, 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.

In another specific embodiment of the present invention, said first module comprises:

- one or more container(s) for accommodating a fluid;

- one or more stirring means for stirring said fluid;

- one or more ports for introducing or removing components in/from said container(s). In a particular embodiment, said second module comprises:

- one or more chromatography columns and/or membranes, which are in fluid connecting with each other;

- optionally one or more filter(s) for protecting said chromatography columns and/or membranes;

- input means for introducing a buffer solution into said chromatography columns and/or onto said membranes;

- output means for removing waste and purified products from said chromatography columns and/or membranes.

In yet another particular embodiment, 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.

In a specific embodiment the present invention said container of the first module of the first bioprocess accommodates a cell culture suspension or adherent cells.

In a particular embodiment, said stirring means of the first module of the first bioprocess is in the form of a cylindrical rotating filter.

In another particular embodiment, 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.

In yet another particular embodiment, 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. In a further aspect, 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.

In a particular embodiment said container of the first module of the second bioprocess accommodates a process fluid containing the protein.

In another particular embodiment, said stirring means of the first module of the second bioprocess is in the form of a magnetic agitator.

In yet another particular embodiment, 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.

In a specific embodiment the present invention, 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.

In a further aspect, the present invention provides a perfusion bioreactor suitable for use as a first module in the system as defined herein.

In a further aspect, the present invention provides a perfusion bioreactor comprising:

- one or more container(s) for accommodating a fluid;

- one or more 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(s); wherein said container is a consumable made from a single-use material, such as plastic. BRIEF DESCRIPTION OF THE DRAWINGS

With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present invention only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the invention. In this regard no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention. The description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

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 Abbreviations: 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. In addition to those 2 main segments (or “modules”), there are other “segments” (or “modules”) serving other process sections: filtration, product storage, waste.

Fig. 3: Implementation of Bioprocess 1 (Panel A) and Bioprocess 2 (Panel B) in the “skid” / “unit”. 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. Abbreviations: DO = dissolved oxygen, IR = infrared spectroscopy. DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

In a first aspect, 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; and

- a second module 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.

In particular, 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; and

- a second module designed to perform chromatography; wherein in operation of said system, said first and second module are in fluid contact with each other thereby allowing to perform sequentially: a) bioprocess 1 comprising cell culturing in the first module and chromatography in the second module; and b) bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.

In another embodiment, 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; and

- a second module designed to perform chromatography; wherein in operation of said system, said first and second module are in fluid contact with each other thereby allowing to perform sequentially: a) bioprocess 1 comprising cell culturing in the first module and chromatography in the second module; and b) bioprocess 2 comprising viral inactivation and/or bioconjugation in the first module and chromatography in the second module.

In a specific embodiment of the present invention, 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.

In a specific embodiment of the present invention, 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.

In the context of the present invention, the term “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. For example, 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).

In the context of the present invention, 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. In the context of the present invention, 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.

In the context of the present invention, 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. In a particular embodiment of the present invention, the perfusion bioreactor as used herein comprises a spin filter, suitable for separating cells from the surrounding liquid medium. In the context of the present invention, 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. In the production of biologies, the most commonly employed inactivation methods are the use of low pH, typically followed by a neutralisation step using a base; or the addition of detergents. Since the efficiency of these processes is clearly linked to the effectiveness of mixing, this step can suitably be performed in the first module (segment A) of the claimed device, which comprises a stirring means.

In the context of the present invention, the term “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 (ADC) 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).

In the context of the present invention, the term “nanofiltration” or “NF” is meant to be a filtration method using membranes and high pressure to remove nanoparticles such as viruses.

In the context of the present invention, the term “ u I traf i I tratio n/d iaf i Itratio n” or “UF/DF” 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.

In the context of the present invention, the term “downstream processing” or “DSP” is meant to be a series of process steps traditionally comprising the second half of the manufacturing process (purification steps). In the context of the present invention, the term “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).

As used herein, the term “bioprocess 1” or “first bioprocess” 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).

As used herein, the term “bioprocess 2” or “second bioprocess” 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.

In the context of the present invention, the term “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. As used herein, 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.

In the context of the present invention, the term “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).

In the context of the present invention, the term “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).

In addition to those 2 main segments or “modules”, there are other “segments” (or “modules”) serving other process sections such as filtration, product storage, waste,... (see Fig. 2). 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. In yet another particular embodiment, 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.

In a further aspect, 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.

In the context of the present invention, the term “modular” is meant to be a modular design wherein each component can be removed and replaced with a similar component. As used herein, 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)

In another specific embodiment of the present invention, said first module of the first bioprocess comprises:

- one or more container(s) for accommodating a fluid;

- one or more stirring means for stirring said fluid;

- one or more ports for introducing or removing components in/from said container(s).

In the context of the present invention, the term “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.

As defined herein, 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.

In the context of the present invention, the term “bioreactor” is meant to be a container and integral vessel suitable for growing cells or micro-organisms. As used herein, 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. CHO, NS0, HEK, other) in perfusion mode. It may also be applicable for adherent cells on a solid support such as microcarriers. 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.

In a specific embodiment the present invention, said container of the first module of the first bioprocess or “bioreactor” accommodates a cell culture suspension or adherent cells.

In the context of the present invention, the term “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.

In a particular embodiment said container of the first module of the second bioprocess or “viral inactivation tank” accommodates a process fluid containing the protein.

In the context of the present invention, the term “process fluid” is meant to be liquid containing the product of interest, which is a protein of therapeutic or diagnostic value.

As used herein, a “stirring means” is referred to as any rotating device acting as a mechanical mixing device to ensure a homogenous mixture. In a specific embodiment, 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. separating 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.

In a specific embodiment, 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. Alternatively, 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.

In a particular embodiment, said stirring means of the first module of the first bioprocess is in the form of a cylindrical rotating filter.

Alternatively, in another particular embodiment, said stirring means of the first module of the second bioprocess (viral inactivation step) 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. This is the same principle of a disc-stack centrifuge and the cells mat be projected by centrifugal force (ii) an in-line depth filter at the outlet of the clarified supernatant for further clarification prior to sterile filtration. 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.

Furthermore, the different modules of the present invention may contain one or more ports.

In another particular embodiment, 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.

In yet another particular embodiment, 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. For example 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.

In the context of the present invention a fluid flow can be made in any of the following methods, or combinations thereof:

• tubing / hoses made of silicone or thermoplastics

• extruded plastic film

• injection moulded parts

• machined parts

• 3D printed parts

Furthermore, a chromatography technique may be performed in the second module of the first bioprocess as well as the second bioprocess.

In a particular embodiment, said second module comprises:

- one or more chromatography columns and/or membranes, which are in fluid connecting with each other;

- optionally one or more filter(s) for protecting said chromatography columns and/or membranes; - input means for introducing a buffer solution into said chromatography columns and/or onto said membranes;

- output means for removing waste and purified products from said chromatography columns and/or membranes.

In the context of the present invention, the term “buffer solution” is meant to be different salt-based solutions used in various steps of the purification processes, as liquid phases.

In yet another particular embodiment, said chromatography columns of the second module of the first bioprocess allow for chromatography such as protein A chromatography capturing.

In a specific embodiment the present invention, said chromatography columns of the second module of the second bioprocess allow for chromatography such as ion exchange chromatography.

In the context of the present invention, the term “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.

In some embodiments, 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.

In the context of the present invention, the term “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.

In the context of the present invention, 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.

- In general, 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

- In this case, 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”

• Whilst the process is intended to run continuously, without interruptions, the system can be rendered more flexible by allowing break bags between different steps. Those break bags help to control the process if, for instance, there is a difference of flow rate between 2 consecutive steps.

In the context of the present invention, the term “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. 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.

In a further aspect, the present invention provides a perfusion bioreactor suitable for use as the first module in the system as defined herein.

In a further aspect, the present invention provides a perfusion bioreactor comprising:

- one or more container(s) for accommodating a fluid;

- one or more 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(s); wherein said container is a consumable made from a single-use material, such as plastic.

EXAMPLES

The different aspects of the modules, segments, bioprocess,... according to the present invention are now further detailed herein below with more specific examples; and where applicable in reference to the figures disclosed herein.

Bioprocess 1 : cell culturing and chromatography purification

Herein below, the different aspects of bioprocess 1 of the present invention are further detailed using some specific examples. Bioreactor Step - Process Control

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.

Spin filter rotation and mixing do not need to be controlled, with the control unit sending a signal to the agitator drive, without feedback. The drive can turn in both directions (clockwise and counterclockwise).

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)

Bioreactor Step - Operation The following bullet-points provide several different aspects of the perfusion bioreactor of the present invention:

• 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

• Once a specific cell density, determined by the user, is reached, 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.

Chromatography Purification - Design In this example a protein A chromatography is used but may also be a technique selected from the list mentioned in the description.

The purification segment of the process may comprise one or more of the following elements:

• A sterile filter to protect the bioreactor from airborne microbial contamination, thus providing a “sterility barrier” between the USP process which is “sterile” and the DSP, which is deemed not be sterile but “low bioburden”

• Additional filters may be added prior to the aforementioned sterility barrier

• 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. Once the 2 parts are disengaged, fluid flow stops and there are no leakages to the outside. 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.

• There could be intermediate product hold steps or break bags between those steps, before or after. This could be required in case of difference of product fluid flow rates between the upstream and downstream processes • 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

Chromatography Purification - Process Control The following parameters may be monitored in the downstream process:

• Process liquid (product) flow rate

• Pressure (TBC)

• Absorbance / UV

Chromatography Purification - Operation The following bullet-points provide several different aspects of the Protein A Purification of the present invention but may (partly) apply for other chromatography techniques as well:

• Prior to use, all columns may be in “equilibrated” mode with the appropriate buffer

• 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 fully loaded column is then washed with wash buffer to remove weaker bound species, which are also referred to as “contaminants”. These are directed to the downstream processing waste bag

• 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

• Loading and elution of different columns is switched between those columns as a result of UV reading of the protein of interest Bioprocess 2 Implementation (Viral Inactivation and Chromatography)

Herein below, the different aspects of bioprocess 2 of the present invention are 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 - Process Control

Viral inactivation is mainly done using pH adjustment.

The following process parameters are monitored and controlled during the viral inactivation step:

• pH of the contents (online) o acid is added progressively during the inactivation step, until the required pH value (around 3.5) is reached o base is then added progressively, during the neutralisation step, until the required pH value is reached (around 6 or as required for the subsequent ion exchange chromatography step)

• the rotation speed of the agitator, which can also be a one-way signal without monitoring and control

Alternatively, samples can be taken from the inactivation vessel, and pre-determined quantity of acid and base are then added.

• In this case, the weight of the vessel content may be monitored and the differential increase measured

Note that this system also allows viral inactivation by detergent using a common detergent used in biopharmaceutical processing, such as Triton-X

In this case a pre-determined volume of detergent, as a proportion of the process fluid, is added

• This can be added based on the following: o A determined pumping time o Measuring the increase in weight in the container of viral inactivation vessel Viral Inactivation - Operation

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.

• Once the required pH is reached, mixing continues for a determined and validated duration which can normally range from about 1 to 4 hours, such as about 1 , about 2, about 3 or about 4 hours.

Chromatography Purification - Design In this example an ion exchange chromatography is used but may also be a technique selected from the list mentioned in the description.

The purification segment of the process may comprise one or more of the following:

• a filter to protect the chromatography columns from aggregates which may have formed during inactivation and any other potential contaminants

• Additional filters may be added if required

• 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

• continuous chromatography can be performed in 2 ways: o using the simulated moving method whereby the flow may 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 (refer to figure 7 in the protein A section above)

• there could be intermediate product hold steps or break bags between those steps, before or after. This could be required in case of difference of product fluid flow rates between the upstream and downstream processes

• 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 columns may be pre-packed with the appropriate resin may comprise the appropriate membrane

Chromatography Purification - Process Control The following parameters may be monitored in the downstream process:

• process liquid (product) flow rate

• pressure (TBC)

• absorbance / UV

Chromatography Purification - Operation The following bullet-points provide several different aspects of the Ion Exchange Purification of the present invention but may (partly) apply for other chromatography techniques as well:

• prior to use, all columns may be brought in “equilibrated” mode with the appropriate buffer

• the process fluid coming out of the viral inactivation step and then into the 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 may be directed to the next column and so on. Any product that breaks through the first column may be directed into the next column, thus minimising product loss.

• the fully loaded column is then washed with wash buffer to remove weaker bound species , which are also referred to as “contaminants”. These are directed to the downstream processing waste bag

• 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.

• loading and elution of different columns may be switched between those columns as a result of UV reading of the protein of interest