Methods and systems for performing perfusion cell culture

Abstract

The present invention relates to methods and systems for performing perfusion cell culture, whereby the supernatant of the effluent stream is recovered.

Description

[0001] The present invention relates to methods and systems for performing perfusion cell culture, whereby the supernatant of the effluent stream is recovered.

[0002] The most commonly used culture modes in biomanufacturing are batch cell culture, fed-batch, and perfusion cell culture. The reason for choosing one of these techniques lies in different factors related to the protein and / or the host. The cells are cultured either attached to a carrier or in suspension. The easiest mode to operate is probably the batch bioreactor. After inoculation, the cells grow and produce until nutrient limitation is reached due to medium consumption and the cell density starts to decrease. The second very common method is fed-batch, where nutrient limitation is prevented by adding highly concentrated feeds at different time points during the culture. Thus, the culture duration is longer than the batch mode, and the final productivity is increased.

[0003] The perfusion cell culture method continuously perfuses fresh medium through the culture, while providing fresh nutrients to the cells and removing the used medium and optionally dead cells and the target product, while retaining a large number of live cells, allowing the bioreactor to operate continuously for long periods of up to several months. The key advantages of the perfusion technique include higher yields per bioreactor volume, increased flexibility, and more consistent product quality. However, to achieve this, the system and method need to be set up very carefully. Different from the fed-batch system, the perfusion system does not accumulate waste. The expressed protein can be quickly removed and used for purification, which is a significant advantage for proteins that are easily unstable.

[0004] Removing the used medium while keeping the cells in culture can be accomplished using different techniques (such as filtration, e.g., alternating tangential flow (ATF) and standard tangential flow filtration (TFF)). Other methods include using sedimentation devices, centrifuges, or acoustic devices. Another option is to retain the cells by binding them to a matrix (capillary fibers, membranes, microcarriers in a fixed bed, etc.) in the bioreactor.

[0005] A review providing details on advantageous setups for perfusion cell culture can be found in "Perfusion mammalian cell culture for recombinant protein manufacturing - A critical review" by Jean-Marc Bielser et al., Biotechnology Advances 36 (2018) 1328 - 1340. A filtration-based perfusion system where dead cells can only be removed from the system by bleed-off is described in "Potential of Cell Retention Techniques for Large-Scale High-Density Perfusion Culture of Suspended Mammalian Cells", D. Voisard, F. Meuwly, P.-A. Ruffieux, G. Baer, A. Kadouri, Cytotechnology 28: 163 - 175, 1998.

[0006] In some perfusion methods, ultrafiltration membranes are used to retain the product in the bioreactor. These methods are also referred to as "concentrated fed-batch" or CFB. Concentrated fed-batch cell culture can increase manufacturing capacity without additional volume capacity. Information on this particular perfusion method can be found in William C. Yanga,∗, Daniel F. Minklera, Rashmi Kshirsagarb, Thomas Ryllb, Yao-Ming Huanga, Journal of Biotechnology 217 (2016) 1 - 11.

[0007] Figure 1 Schematic diagram showing a prior art perfusion cell culture bioreactor. A bioreactor (1) having a cell culture (2) comprising a liquid cell medium and cells is optionally agitated by a stirrer 3. Fresh new medium can be added via Q-inlet (also referred to as P). A harvest stream comprising cells, liquid medium, and the target product exits the bioreactor (1) via the Q-harvest line. Q-harvest is typically referred to as H. A cell retention device (4) retains the cells, for example, by the methods described above, such that a cell-free or cell-reduced harvest can be collected. Generally, in perfusion cell culture, the medium is continuously fed via Q-inlet, while the harvest is continuously withdrawn via Q-harvest.

[0008] Once the cell density reaches the desired set point, it is necessary to remove the excess cells to maintain a stable cell concentration and achieve steady-state operation. This is accomplished via the effluent stream Q-effluent (also referred to as B). This stream consists of a liquid and a solid fraction, which is a suspension. The solid fraction includes live and dead cells, and the liquid fraction includes the liquid cell culture medium as well as waste components and the target product present in the liquid. To maintain a constant volume in the bioreactor, generally Q-in = Q-harvest + Q-effluent, also known as P = H + B, meaning that the volume of cell culture medium newly added to the bioreactor via Q-in needs to be equal to the volume removed via Q-harvest and Q-effluent.

[0009] As discussed, for example, in Jean-Marc Bielser et al., Biotechnology Advances 36 (2018) 1328-1340, this effluent stream is wasted without recovering the target product. Therefore, to maximize the process yield, generally the effluent stream rate is minimized.

[0010] However, the performance and yield of the process depend on the different flow rates. An increased perfusion rate generally enables the production of more biomass and thus more of the target product. The faster the cells grow, the greater the effluent rate leading to a yield loss. Therefore, steady-state operation is typically defined within a range where the cell density is high enough to achieve an economically viable productivity, but where cell growth is nutrient-limited or controlled by other environmental factors to minimize the effluent rate.

[0011] Therefore, it would be advantageous to find methods that allow not only an economic cell density but also a high, more productive cell density. It has been found that this can be achieved by inserting an effluent recovery device into the bioreactor system. With this device, the effluent, which needs to be removed from the bioreactor to remove excess live and dead cells but always still contains the target product, is first separated into a solid fraction mainly containing cells and a liquid fraction containing the liquid with the target product. Then the liquid fraction can be directed back into the bioreactor or it can be directed into the harvest stream. In this way, the major part of the target product present in the effluent is not wasted but can be recovered. This results in a higher process yield and allows additional degrees of freedom in process optimization, since there is no longer a need to limit the effluent stream to reduce the loss of the target product. If this is beneficial for a higher product yield, the effluent stream can instead be increased.

[0012] Accordingly, the present invention relates to a bioreactor system which comprises a bioreactor having a culture medium inlet and a harvest outlet and generally includes a cell retention means whereby the bioreactor additionally includes an effluent recovery means having an inlet for the effluent, means for separating the cells of the effluent from the liquid portion of the effluent and an outlet, the inlet directing the effluent from the bioreactor to the means for separating the cells of the effluent from the liquid portion of the effluent and the outlet for directing the liquid portion of the effluent into the bioreactor and / or the harvest. The cells accumulate in the effluent recovery means over time and over several cycles. Accordingly, a flexible container is preferred. A cell slurry can also be removed between cycles.

[0013] In a preferred embodiment, the means for separating the cells of the effluent from the liquid portion of the effluent is a container or a bag.

[0014] In a preferred embodiment, the system additionally includes a pump for controlling the flow of the effluent into the effluent recovery means and in particular into the means for separating the cells of the effluent from the liquid portion of the effluent, and a pump for controlling the flow of the liquid portion of the effluent out of the means for separating the cells of the effluent from the liquid portion of the effluent through the outlet of the effluent recovery means back into the bioreactor and / or into the harvest outlet.

[0015] In the simplest case, a line or a tube, a pump and a container are sufficient to effect effluent recovery. However, additional tubes and pumps can increase the efficiency of the effluent recovery means. In one embodiment, there are two separate settling containers which are used alternately, which allows quasi-continuous operation.

[0016] In a preferred embodiment, the system additionally includes a process management system.

[0017] In a preferred embodiment, the inlet of the effluent recovery means is a tube, preferably a sealable plastic tube. The sealing of the tube can be accomplished by a pump, by a valve or by other locking mechanisms.

[0018] In a preferred embodiment, the outlet of the effluent recovery means is a tube, preferably a sealable plastic tube. The sealing of the tube can be accomplished by a pump, by a valve or by other locking mechanisms.

[0019] In a preferred embodiment, the outlet of the effluent recovery means leads into the bioreactor. Preferably, it enters the bioreactor at a submerged position (which means a position below the surface of the culture medium and the cell suspension).

[0020] In another preferred embodiment, the means for separating the cells of the effluent from the liquid portion of the effluent is a container for settling the cells.

[0021] In a preferred embodiment, the outlet of the effluent recovery device is connected to a container for sedimenting cells at the opposite side to the connection of the inlet and to the upper half of said side.

[0022] In a highly preferred embodiment, the container for sedimenting cells is a flat container, the minimum dimension of which is the height when it is placed horizontally.

[0023] In a highly preferred embodiment, the flat container is a plastic bag.

[0024] The present invention also relates to a method for perfusing cell cultures, which comprises culturing cells in a bioreactor system, said system comprising a bioreactor having a medium inlet and a harvest outlet and an effluent recovery device having an inlet for effluent (for guiding effluent from the bioreactor to a device for separating the cells from the liquid part of the effluent) and an outlet (for guiding the liquid part of the effluent from the device for separating the cells from the liquid part of the effluent back into the bioreactor or into the harvest outlet), whereby

[0025] i. continuously or one or more times during the cell culture process, inserting fresh cell culture medium into the bioreactor via the medium inlet

[0026] ii. continuously or one or more times during the cell culture process, removing harvest from the bioreactor via the harvest outlet

[0027] iii. continuously or one or more times during the cell culture process, removing a quantity of effluent from the bioreactor via the inlet of the effluent recovery device and guiding it to a device for separating the cells from the liquid part of the effluent, separating the cells and the liquid part, and transferring the liquid part back into the bioreactor and / or into the harvest outlet via the outlet of the effluent recovery device.

[0028] In a preferred embodiment, in iii, the liquid part of the effluent transferred back into the bioreactor and / or into the harvest outlet contains more than 70%, preferably more than 80%, of the liquid contained in the effluent removed from the bioreactor and less than 10%, preferably less than 5%, for example between 1 - 5%, such as about 1%, about 2%, about 3% or about 4%, of the cells contained in the effluent removed from the bioreactor.

[0029] In a preferred embodiment, iii is carried out one or several times during the cell culture process. A certain amount of the effluent is taken out from the bioreactor through the inlet of the effluent recovery device and guided to a device including a container for separating the cells in the effluent from the liquid part of the effluent. The effluent is allowed to settle in the container for a period of time such that the cells settle towards the bottom surface of the container. Thereafter, the cell-reduced liquid supernatant above the settled cells is taken out through the outlet of the effluent recovery device and returned to the bioreactor and / or introduced into the harvest outlet.

[0030] In a preferred embodiment, the time for allowing the effluent to settle in the container is between 30 minutes and 2 hours.

[0031] In a preferred embodiment, the method steps i, ii and iii are adjusted such that the volume of the cell culture in the bioreactor is maintained at a constant level. In addition, by advanced control of the flow rates B and H, P can be kept constant, which is considered a prerequisite for steady-state perfusion operation.

[0032] A schematic diagram of a perfusion bioreactor system according to the prior art can be seen in Figure 1 . A schematic diagram of a perfusion bioreactor system including an effluent recovery device according to the present invention is shown in Figure 2 .

[0033] Figure 3 Shows the viable cell density of the liquid part of the effluent re-fed into the bioreactor from the effluent recovery device after cell sedimentation.

[0034] Figure 4 Shows an exemplary flow rate profile of a perfusion method according to the present invention with 2 vvd.

[0035] Regarding Figures 5 - 11 Details can be seen in the examples.

[0036] Cell culture is any setting in which cells are cultured.

[0037] Cell culture is generally carried out in a bioreactor.

[0038] A bioreactor is any container suitable for cell culture, such as a bottle, tube, vessel, bag, flask and / or tank. Generally, the container is sterilized before use. Cell culture is generally carried out by incubating the cells in an aqueous cell culture medium under conditions suitable for cell growth and / or maintenance (such as suitable temperature, pH, osmolarity, aeration, agitation, etc.), which conditions limit contamination by foreign microorganisms from the environment. Those skilled in the art are aware of the suitable incubation conditions for culturing cells. The bioreactor used according to the present invention is preferably a bioreactor suitable for perfusion cell culture.

[0039] The bioreactor system suitable for the present invention comprises a bioreactor and additional equipment necessary for running perfusion cell culture in the bioreactor, such as one or more of the following

[0040] - Means for agitation

[0041] - Means for supplying components to and discharging components from the bioreactor, such as pipes, pumps, valves, storage tanks

[0042] - Cell retention means (see above)

[0043] - A system for monitoring the volume of the bioreactor, such as a bioreactor balance, a liquid level sensor, etc.

[0044] - Means for controlling and maintaining temperature, osmolarity, aeration, agitation, etc.

[0045] - A computer system for automated or semi-automated operation of a cell culture bioreactor.

[0046] A cell culture medium according to the present invention (synonymously used: culture medium) is any mixture of components for maintaining and / or supporting cell growth in vitro and / or supporting or maintaining a specific physiological state.

[0047] It may contain undefined components, such as plasma, serum, embryo extract or other undefined biological extracts or peptones. Preferably, it may also be a chemically defined medium. A cell culture medium may contain all components necessary for maintaining and / or supporting cell growth in vitro or selected components (medium supplements) for addition, either in combination or not, with other components added separately. The components of a cell culture medium are also referred to as cell culture medium ingredients.

[0048] The cell culture device and method according to the present invention are designed to be suitable for growing prokaryotic cells (such as bacterial cells) as well as eukaryotic cells (such as yeast, fungi, algae, plants, insects and / or mammalian cells) and optionally archaea or for maintaining / supporting their growth. Preferred cells are mammalian cells.

[0049] A chemically defined cell culture medium is a cell culture medium containing 'defined' raw materials that are well characterized chemically. This means that the chemical composition of all the chemicals used in the medium is known. A chemically defined medium does not contain chemically undefined substances, such as chemically undefined yeast, animal or plant tissues; they do not contain peptones, feeder cells, serum, undefined extracts or digests or other components that can contribute proteins and / or peptides and / or hydrolysis products that are chemically less defined to the medium. In some cases, a chemically defined medium may contain chemically defined proteins or peptides, an example being insulin (see other below).

[0050] Liquid cell culture media are generally produced by dissolving powdered cell culture media in a suitable liquid.

[0051] Powdered cell culture media, dry powder media, or dehydrated media are cell culture media generally produced by a grinding method or a freeze-drying method. This means that powdered cell culture media are generally fine-grained particulate media, rather than liquid media. The term "dry powder" may be used interchangeably with the term "powder"; however, unless otherwise specified, "dry powder" as used herein refers only to the overall appearance of the particulate material and is not intended to mean that the material is completely free of combined or aggregated solvent. Powdered cell culture media may also be granular cell culture media, for example, produced by dry granulation by roller compaction or wet granulation by fluidized bed spray granulation. Such media may also be prepared by spray drying.

[0052] The solvent (also referred to as the liquid) used to prepare liquid cell culture media is generally water (most particularly distilled and / or deionized water or purified water or water for injection or water purified by reverse osmosis (Milli-Q®)) or an aqueous buffer. The solvent may also contain saline, soluble acid or base ions that provide a suitable pH range (generally in the range of pH 1 - pH 10), stabilizers, surfactants, preservatives, and alcohols or other polar organic solvents.

[0053] Before adding cells, the pH of the dissolved media is generally between pH 2 - 12, more preferably between pH 4 - 10, even more preferably between pH 6 - 8, and most preferably between pH 6.5 - 7.5, and ideally between pH 6.8 - 7.3.

[0054] Cell culture media that contain all the components necessary to maintain and / or support the growth of cells in vitro generally contain at least one or more sugar components, one or more amino acids, one or more vitamins or vitamin precursors, one or more salts, one or more buffer components, one or more cofactors, and one or more nucleic acid components (nitrogenous bases) or their derivatives. It may also contain chemically defined biochemicals, such as recombinant proteins, for example, rInsulin, rBSA, rTransferrin, rCytokine, etc.

[0055] The culture medium may also contain sodium pyruvate, highly purified and thus chemically well-defined extracts, fatty acids and / or fatty acid derivatives, and / or poloxamer product components (block copolymers based on ethylene oxide and propylene oxide), in particular poloxamer 188 sometimes referred to as Pluronic F 68 or Kolliphor P 188 or Lutrol F 68, and / or surface-active components, such as chemically prepared non-ionic surfactants. An example of a suitable non-ionic surfactant is a bifunctional block copolymer surfactant with a primary hydroxyl group at the end, also known as a poloxamer, which can be obtained, for example, from BASF, Germany under the trade name pluronic®. Such poloxamer product components are hereinafter only referred to as poloxamer or pluronic. Chelating agents, hormones and / or growth factors may also be added.

[0056] Other components it may contain are lactic acid, mercaptoacetic acid, thiosulfate, tetrathionate, diaminobutane, inositol, phosphatidylcholine (lecithin), sphingomyelin, iron-containing compounds (including compounds with iron-sulfur clusters), uric acid, carbamyl phosphate, succinic acid, thioredoxin, orotic acid, phosphatidic acid, polyamines (such as putrescine, spermidine, spermine and / or cadaverine), triglycerides, steroids (including but not limited to cholesterol), metallothionine, oxygen, glycerol, urea, α-ketoglutarate, ammonia, glycerol phosphate, starch, glycogen, glyoxylate, isoprenoids, methanol, ethanol, propanol, butanol, acetone, lipids (including but not limited to those in micelles), tributyrin, butyrin, cholate, deoxycholate, polyphosphates, acetate, tartrate, malate and / or oxalate, in the form of pure compounds, salts, conjugates and / or derivatives.

[0057] The sugar components are all monosaccharides or disaccharides, such as glucose, galactose, ribose or fructose (examples of monosaccharides) or sucrose, lactose or maltose (examples of disaccharides) or their derivatives such as sugar alcohols. The sugar components may also be oligosaccharides or polysaccharides.

[0058] Examples of amino acids according to the invention are in particular protein amino acids, especially essential amino acids, leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine, and non-protein amino acids, such as D-amino acids.

[0059] Tyrosine means L- or D-tyrosine, preferably L-tyrosine.

[0060] Cysteine means L- or D-cysteine, preferably L-cysteine.

[0061] Amino acid precursors and analogues are also included.

[0062] Examples of vitamins are vitamin A (retinol, retinal, various retinols, and 4 carotenoids), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin, nicotinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxol, pyridoxamine, pyridoxal), vitamin B7 (biotin), vitamin B9 (folic acid, folinic acid), vitamin B 12 (cyanocobalamin, hydroxocobalamin, methylcobalamin), vitamin C (ascorbic acid) (including phosphates of ascorbic acid), vitamin D (ergocalciferol, cholecalciferol), vitamin E (tocopherols, tocotrienols), and vitamin K (phylloquinone, menaquinone). Also included are vitamin precursors and analogs.

[0063] Examples of salts are components containing inorganic ions such as bicarbonate, calcium, chloride, magnesium, phosphate, potassium, and sodium or trace elements such as Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn. Examples are copper(II) sulfate pentahydrate (CuSO4 . 5 H2O), sodium chloride (NaCl), calcium chloride (CaCl2 . 2 H2O), potassium chloride (KCl), iron(II) sulfate, anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous magnesium sulfate (MgSO4), anhydrous disodium hydrogen phosphate (Na2HPO4), magnesium chloride hexahydrate (MgCl2 . 6H2O), zinc sulfate heptahydrate (ZnSO4 . 7 H2O).

[0064] Examples of buffers are carbonates, citrates, phosphates, HEPES, PIPES, ACES, BES, TES, MOPS, and TRIS.

[0065] Examples of cofactors are thiamine, biotin, vitamin C, calciferol, choline, NAD / NADP (reduced and / or oxidized), cobalamin, vitamin B12, flavin mononucleotide and derivatives, flavin adenine dinucleotide and derivatives, glutathione (reduced and / or oxidized and / or as a dimer), heme, hemin, hemoglobin, ferritin, nucleotide phosphates and / or derivatives (e.g., adenosine phosphate), coenzyme F420, s-adenosylmethionine, coenzyme B, coenzyme M, coenzyme Q, acetyl coenzyme A, molybdopterin, pyrroloquinoline quinone, compounds, salts, complexes, and / or derivatives of tetrahydrobiopterin.

[0066] Nucleic acid components are nucleobases (such as cytosine, guanine, adenine, thymine, uracil, xanthine and / or hypoxanthine), nucleosides (such as cytidine, uridine, adenosine, xanthosine, inosine, guanosine and thymidine) and nucleotides (such as adenosine monophosphate or adenosine diphosphate or adenosine triphosphate), including but not limited to their deoxy and / or phosphate ester derivatives and / or dimers, trimers and / or polymers, such as RNA and / or DNA.

[0067] Additive components can be added, which improve the physicochemical properties of the culture medium, for example but not limited to increasing the clarity and / or solubility of the culture medium and / or one or more of its components, without significantly negatively affecting the cell growth characteristics at the concentrations used. Such components include but not limited to chelating agents (such as EDTA), antioxidants, detergents, surfactants, emulsifiers (such as polysorbate 80), neutralizing agents (such as polysorbate 80), micelle formers, micelle inhibitors and / or polypropylene glycol, polyvinyl alcohol and / or carboxymethyl cellulose.

[0068] As used herein, the terms "cell density", "viable cell density" and "cell concentration" can be interchangeably used to refer to the number of metabolically active cells per unit volume of cell culture.

[0069] The term "perfusion" or "perfusion method" refers to a cell culture method for producing a target product (such as an antibody or a recombinant protein), in which a high cell concentration in a bioreactor continuously or one or more times receives fresh growth medium during cell culture, whereby the used medium that may contain the target product is harvested, which means that it is continuously or one or more times removed from the bioreactor during cell culture. Preferably, fresh growth medium is continuously fed into the bioreactor, and the used medium that may contain the target product is continuously harvested.

[0070] Cells to be cultured with the systems and methods of the present invention can in particular be cells capable of expressing a target product, such as a therapeutic biomolecule, such as an immunoglobulin (such as a monoclonal antibody or an antibody fragment), a fusion protein, a clotting factor, an interferon, insulin, a growth hormone or other recombinant proteins. Such cells can be, for example, CHO cells, baby hamster kidney (BHK) cells, PER.C.6 cells, myeloma cells, HEK cells, etc.

[0071] "Steady state" generally refers to a stable state that does not change over time or in which a change in one direction is continuously balanced by a change in the other direction. In perfusion, the steady state can be defined by a "constant viable cell density". The combination of a constant viable cell density and a constant perfusion rate results in a constant cell-specific perfusion rate (CSPR), which is generally considered to be the key criterion for achieving a steady state.

[0072] Generally, for perfusion cell culture, a small amount of cells and a liquid cell culture medium are introduced into a bioreactor and culture conditions are selected such that the cells divide and thus an increased cell density is produced while expressing the target product. The culture can be carried out according to methods known in the art, involving, for example, an appropriate degree of agitation, addition of oxygen / air, removal of CO2 and other gaseous metabolites, etc. During the culture, various parameters such as pH, conductivity, metabolite concentration, cell density, etc. can be controlled to provide conditions suitable for a given cell type. The cell density can be appropriately increased to a level where the cell concentration in the bioreactor is at least 1 million cells / ml, preferably at least 10 million cells / ml, generally between 10 million - 250 million cells / ml. The upper limit will be mainly determined by the rheological properties of the cell suspension at very high cell densities, where agitation and gas exchange are hindered when approaching a paste-like consistency. Other limitations of the perfusion method may prevent the operator from reaching this physical limit, such as the maximum cell retention device flow rate, the maximum bioreactor oxygen transfer rate, product stability limitations, and the minimum cell-specific perfusion rate (CSPR) allowed by the culture medium. The cell viability can be, for example, at least 50%, such as at least 80% or at least 90%.

[0073] The concentration of the target product expressed by the cells in the bioreactor can be at least 0.1 g / l or at least 2 g / l. Generally, it is between 0.1 - 5 g / l, but in some methods such as CFB (where the product is not harvested but retained in the bioreactor), a product concentration of up to 10 - 30 g / l can be achieved.

[0074] Exemplary bioreactors suitable for perfusion cell culture include cell retention devices to keep the cells in the bioreactor during harvesting. Such cell retention devices can be acoustic, alternating tangential flow (ATF), settlers, centrifuges, etc. In some instances, disposable, reusable, or semi-disposable bioreactors can be used. Any combination of hardware designs can be used. In one instance, a disposable cell retention device can be used. In some embodiments, disposable conduits, tubing, pumps, bag assemblies, and cell retention devices are used instead of hard tubing and reusable devices.

[0075] The bioreactor of the bioreactor system of the present invention can have any suitable volume, including but not limited to about 1 L - about 2000 L, but the volume is not limited to this exemplary range. Some exemplary bioreactor volumes include but are not limited to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 500, 1000, 1500 L, any intermediate volume, etc.

[0076] Exemplary bioreactors can have any suitable minimum and maximum working volumes, depending on, for example, the total vessel volume, the ratio between the height and diameter of the vessel, the vessel configuration (e.g., whether the bioreactor is a bag bioreactor), the growth rate, etc. For example, in a 5 L bioreactor, the exemplary minimum working volume can be in the range of about 100 mL - about 1 L, and the exemplary maximum working volume can be in the range of about 3.5 L - about 5 L. In a 20 L bioreactor, the exemplary minimum working volume can be in the range of about 100 mL - about 5 L, and the exemplary maximum working volume can be in the range of about 15 L - about 19 L. In a 200 L bioreactor, the exemplary minimum working volume can be in the range of about 20 mL - about 50 L, and the exemplary maximum working volume can be in the range of about 150 L - about 190 L. Those of ordinary skill in the art will recognize that the above values and ranges are illustrative and not intended to limit the scope of the present invention.

[0077] The bioreactor can include one or more inlets, also referred to as inlet ports, for introducing one or more feeds (e.g., cell culture medium), chemicals (e.g., pH buffer), antifoaming agents, etc. It can also include one or more outlets, also referred to as outlet ports, for removing cells and / or liquids from the bioreactor. Any suitable means for initiating and directing the flow of fluid through the inlets and / or outlets can be provided to each inlet and / or outlet of the bioreactor, including but not limited to one or more peristaltic pumps, one or more pressurizing means, etc. Any suitable means for monitoring and controlling the flow of fluid through the inlets can be provided to each inlet and / or outlet, including but not limited to one or more mass flow meters, one or more flow control valves, etc. For example, the bioreactor can include a flow control mechanism to control the flow rate of substances in and out of the bioreactor.

[0078] The bioreactor can also include means for volume and / or level control.

[0079] The bioreactor includes a culture medium inlet, which can be operated either discretely or continuously to introduce fresh cell culture medium into the cell culture. The bioreactor includes one or more harvest outlets for discharging the used cell culture, cells, and / or target product. The harvest outlet may include a flow control valve to control the rate of harvest. In one embodiment, the harvest can be stored in a harvest bottle or container.

[0080] The bioreactor system of the present invention further includes an effluent recovery device. A flow control valve can be provided at its inlet to control the effluent rate and the duration of effluent extraction.

[0081] In one embodiment, the device (6) of the bioreactor and / or the effluent recovery device ( Figure 2 ) is a flexible non-porous plastic bag, such as made of a flexible polymer (e.g., polyethylene material or film). Generally, fittings are attached to the bag. As used herein, the term "fitting" refers to a separate object that is welded (e.g., heat welded) to the non-porous bag film for attachment. Thus, the fitting generally comprises a polymeric material that is the same or similar to the polymeric material constituting the wall of the non-porous bag. The fitting is generally a material that is denser than the wall of the non-porous bag and can be added to the bag to achieve functionality. Non-limiting examples of fittings are fittings that form an inlet or an outlet. In a preferred embodiment of the present invention, the non-porous bag includes at least one inlet and outlet in the non-porous bag wall to add or remove liquid or cells from the bag, whereby it can be a combined inlet and outlet or a separate inlet and a separate outlet. The bag may also include more than one inlet and / or more than one outlet. In the case of the device (6) of the effluent recovery device, the bag preferably includes one inlet and one or two outlets.

[0082] Generally, pipes are attached to the inlet and / or outlet.

[0083] In various embodiments of the present invention, the non-porous bag of the effluent recovery device is a two-dimensional disposable bag including a top plate and a bottom plate, or a three-dimensional disposable bench-top bioreactor bag, or a disposable bioreactor bag used with a support structure. The non-porous bag can be of any size, for example having an internal volume of 1 liter, 10 liters, 100 liters, 200 liters, 500 liters, or 5000 liters.

[0084] The pipes are generally flexible or non-flexible pipes made of plastic or metal. Preferably, they are flexible plastic pipes. The diameter and length of the pipes depend on the size of the bioreactor system. Generally, the inner diameter of the pipes is between 2 - 50 mm, generally between 2 - 30 mm.

[0085] Generally, the bioreactor system further includes a pump and a valve connected by a pipe. The pump is used to transport a liquid or suspension or cell slurry from the bioreactor to, for example, a harvest or effluent recovery device or to transport a liquid or suspension or cell slurry from, for example, a harvest or effluent recovery device to the bioreactor or other locations. Examples of suitable pumps are peristaltic pumps, magnetic coupling pumps, diaphragm pumps, etc.

[0086] The position of the valve is such that it can obstruct, permit, or direct the flow of, for example, a fluid, cell suspension, or cell slurry. Examples of suitable valves are, for example, solenoid valves or pinch valves.

[0087] The bioreactor system may include one or more sensors or probes for real-time detection of one or more operating parameters, including but not limited to the status of the inlet port, the status of the outlet port, the status of the manifold, capacitance probes, cell culture volume sensors, cell culture bioreactor weight sensors, liquid level sensors, thermometers, pH probes, oxygen probes, lactate probes, ammonia probes, agitation rate sensors, metabolic flux sensors, metabolic rate sensors, perfusion rate sensors, carbon monoxide sensors, mass spectrometry, gas chromatography, combinations thereof, etc. These sensors can detect one or more operating parameters, including but not limited to viable cell density (using capacitance probes or any alternative method providing on-line measurement of cell density), cell culture volume, cell culture weight, cell culture liquid level, temperature, pH, dissolved oxygen, agitation rate, metabolic flux, metabolic rate, perfusion rate of the perfusion device, oxygen uptake rate, carbon dioxide production (e.g., using gas chromatography, mass spectrometry), lactate level, ammonia level, combinations thereof, etc. The bioreactor may also include soft sensors.

[0088] The bioreactor and its inlet port, outlet port, etc. can be coupled to one or more process management systems, which are configured or programmed to perform multivariate analysis of sensor data and automatically control the operation of the bioreactor in real time based on the analysis. The process management system can control the operation by, for example, opening / closing the ports of the inlet or outlet, changing the status of the manifold, changing the perfusion rate of the bioreactor system, changing the agitation rate, temperature, pH, dissolved oxygen level, combinations thereof, etc. of the cell culture.

[0089] The effluent recovery device includes an inlet for the effluent (to direct the effluent from the bioreactor into a device for separating the cells of the effluent from the liquid portion of the effluent) and an outlet for the liquid portion of the effluent (to direct from the device for separating the cells of the effluent from the liquid portion of the effluent to the bioreactor and / or the harvest outlet). In this case, the harvest outlet means any part of the harvest outlet, such as a pipe or a harvest container. The effluent recovery device can be made of soft or hard materials (such as metal or preferably plastic) forming a defined, closed, sterile volume.

[0090] A schematic view of a perfusion bioreactor system including an emissions recovery device according to the present invention is shown in Figure 2 .

[0091] A bioreactor (1) having a cell culture (2) comprising a liquid cell culture medium and cells is optionally agitated by a stirrer (3). Fresh new medium can be added via Q-inlet. A harvest stream comprising cells, liquid medium, and a target product exits the bioreactor (1) via the Q-harvest line. A cell retention device (4) retains cells, for example by the above-described methods, such that a cell-free harvest can be collected and the cells are retained in the bioreactor. Emissions to be removed from the bioreactor are extracted therefrom via Q-emission and directed via an inlet (5) into a device (6) for separating the cells of the emissions from the liquid portion of the emissions, the inlet directing the emissions from the bioreactor into the device (6). In this device (6), the cells of the emissions are separated from the liquid portion of the emissions, for example by sedimentation, acoustic means, filtration, or centrifugation, preferably by sedimentation. In this case, sedimentation means separating a suspension of cells into a concentrated cell slurry and a cell-free or cell-reduced supernatant by allowing the cells to sediment due to gravity. The supernatant, which generally contains liquid cell culture medium, waste, and target product, is removed from the device (6) via an outlet (7). Then, it can be directed back into the bioreactor (according to route a) or directed to the harvest outlet (according to route b). For emissions recovery, route a) is of course preferred. Optionally, the device (6) for separating the cells of the emissions from the liquid portion of the emissions further includes an outlet (8) through which the cell slurry can be removed and transferred to waste. In a preferred embodiment, the position of the outlet (7) for the liquid is such that only the liquid supernatant is removed through this outlet. This is accomplished, for example, by positioning the height of the outlet such that it is above the height of the sedimented cells. The outlet (7) can also include a filter or equivalent means to prevent cells from flowing through the outlet (7). Preferably, the outlet (7) is located as far as possible from the inlet (5) in the device (6) and at a position in the upper half of the device (6) such that when the liquid supernatant is removed from the device (6) through the outlet (7), the sedimented cells are not disturbed by the liquid flow or carried away by the liquid flow. Preferably, the outlet (7) is directed back into the bioreactor.

[0092] On the other hand, the outlet (8) is an optional outlet designed to remove the sedimented cells. After each separation of the discharge portion into a liquid supernatant portion and cells, or once the amount of sedimented cells in the device (6) is such that further suspension from the bioreactor cannot be inserted into the device (6), the sedimented cells are removed from the device (6) via the outlet (8). Preferably, the outlet (8) is located at the bottom of the device (6) to facilitate removal of the cell slurry by gravity. Of course, the cells can also be removed by other means via an outlet at a different location. The discharge recovery device may also include a sensor or probe for determining the cell density. The cell density at the inlet is generally the cell density of the bioreactor, but the cell density at the outlet is much lower because ideally as few cells as possible should be transferred back to the bioreactor. The cell density sensor can indicate whether the discharge recovery device may be too full or the sedimentation of the cells is not working properly, such that too many cells are transferred back to the bioreactor.

[0093] The inlet (5) and the outlets (7) and (8) generally comprise pipes with valves and / or pumps. The inlet and the outlets are preferably located in a submerged position in the bioreactor.

[0094] In one embodiment, the discharge recovery device comprises two devices (6) each independently connected to the inlet and the outlets. It generally also includes at least one pump or valve that can direct the suspension flow from the bioreactor through the inlet into one of the devices (6) and direct the liquid supernatant from one of the devices (6) back into the bioreactor and / or into the harvest. If there are two or more devices (6), the discharge recovery can be carried out by quasi - continuously and alternately loading two or even more devices (6). When one device (6) is loaded with a fresh cell suspension from the bioreactor, the cells in the other device (6) can sediment, and then the liquid supernatant can be directed back into the bioreactor and / or into the harvest.

[0095] In one embodiment, the inlet and the outlet of the discharge recovery device are the same. In this case, which is the simplest setup, the discharge recovery device is connected to the bioreactor via only one pipe that serves as both the inlet and the outlet. Pumps and / or valves are used to direct the flow. First, the pump pumps the discharge from the bioreactor into the discharge recovery device. Then the cells are allowed to sediment in the container (device 6) of the discharge recovery device. After that, the liquid supernatant is pumped back into the bioreactor. This can be done several times until the cells in the discharge recovery device container are too full to allow effective sedimentation of the cells and removal of the supernatant.

[0096] In the case as above, when the discharge recovery device is meant to be filled with fresh discharge several times without removing the sedimented cells, a flexible plastic bag is the preferred type of container.

[0097] The emissions recovery device of the present invention can be distinguished from other inlet or outlet systems such as harvesting by the outlet leading back into the bioreactor. The outlet may also lead to harvesting, but very preferably, one outlet leads back into the bioreactor. In a preferred embodiment, the emissions recovery device is not connected to any other inlets and outlets or pipelines of the bioreactor system, but it only has inlets and outlets leading into and out of the bioreactor, and preferably it has at least one pump for guiding the flow into and out of the emissions recovery device.

[0098] The present invention also relates to a method for perfusion cell culture, which comprises culturing cells in a bioreactor system, the system comprising a bioreactor having a culture medium inlet and a harvest outlet, and an emissions recovery device having an inlet for emissions (for guiding emissions from the bioreactor to a device for separating the cells from the liquid part of the emissions) and an outlet (for guiding the liquid part of the emissions from the device for separating the cells from the liquid part of the emissions back into the bioreactor and / or into the harvest outlet), whereby

[0099] i. continuously or one or more times during the cell culture process, inserting fresh cell culture medium into the bioreactor through the culture medium inlet

[0100] ii. continuously or one or more times during the cell culture process, removing the harvest from the bioreactor through the harvest outlet

[0101] iii. continuously or one or more times during the cell culture process, removing a certain amount of emissions from the bioreactor through the inlet of the emissions recovery device and guiding it to a device for separating the cells from the liquid part of the emissions, separating the cells from the liquid part, and transferring the liquid part back into the bioreactor and / or into the harvest through the outlet of the emissions recovery device.

[0102] With a bioreactor system comprising an emissions recovery device, perfusion cell culture can be carried out more flexibly. In particular, by removing emissions without product loss, the amount of emissions removed from the bioreactor can be freely selected without having to limit it to a minimum to reduce product loss.

[0103] In a preferred embodiment, step iii is carried out by pumping a determined amount of the cell suspension through an inlet from the bioreactor into a device for separating the cells of the effluent from the liquid part of the effluent. In the case where the device provides separation of the cells from the liquid by sedimentation, after stopping the pumping into the device for separating the cells of the effluent from the liquid part of the effluent, sedimentation begins and the cells sediment and move towards the bottom of the device. Once the cells have sedimented and preferably a solid cake has formed at the bottom of the device, the liquid supernatant can be removed and fed back into the bioreactor or can be added to the liquid harvest. Generally, the cells are allowed to sediment for 30 minutes to 5 hours, preferably 30 minutes to 2 hours. Sedimentation can also be carried out shorter / faster, i.e., less than 30 minutes, depending on the desired degree of cell separation. For some processes, it may be sufficient to remove some of the cells in the effluent if this increases flexibility.

[0104] Generally, the amount of liquid fed back into the bioreactor or into the harvest is more than 50% of the volume of the effluent cell suspension that has been removed from the bioreactor and pumped into the device for separating the cells of the effluent from the liquid part of the effluent. More preferably, it is more than 70 vol%, and most preferably it is between 75 - 90 vol% that is re-fed into the bioreactor or added to the harvest. Preferably, it is re-fed into the bioreactor.

[0105] Preferably, the liquid part of the effluent re-fed into the bioreactor or added to the harvest does not contain any cells. This can be achieved by inserting a cell retention device (such as a filtration device, an acoustic device, etc.) similar to the device used at the harvest outlet into the effluent recovery device. Especially if the liquid part of the effluent is re-fed into the bioreactor, it is not mandatory for it to be absolutely cell-free. Thus, when referring to the liquid part of the effluent, it means the part of the effluent that has at least a reduced cell count compared to the effluent that has been removed from the bioreactor and directed into the effluent recovery device. Ideally, it contains less than 10%, preferably less than 5%, and most preferably less than 2%, for example about 1%, of the viable cell density in the effluent that has been removed.

[0106] Figure 3 Shows the viable cell density of the liquid part of the effluent re-fed into the bioreactor from the effluent recovery device after cell sedimentation. It can be seen that the VCD remains constant until more than 80% of the liquid part of the effluent has been re-fed.

[0107] Preferably, the device for separating the cells of the effluent from the liquid part of the effluent is a flat container, the smallest dimension of which is its height. Preferably, the height is less than half of either of the other two dimensions, and most preferably less than a quarter. The other two dimensions, width and depth, can be selected according to the amount of effluent to be recovered. Generally, this depends on the size of the bioreactor. Dimensions between 10 cm and 100 cm are suitable.

[0108] Although the device for separating the cells of the effluent from the liquid part of the effluent preferably functions by separating the liquid supernatant and the cells by sedimentation, other devices can also be used. Such devices that can also be used for separating cells and liquid harvests can be used, such as filtration, centrifugation, acoustic devices, etc.

[0109] Generally, the whole process is run according to the common process requirements of perfusion cell culture. Preferably, the perfusion rate is kept constant such that the perfusion rate (addition of fresh cell culture medium) (P) = the effluent rate (B) + the harvest rate (H), thereby achieving steady-state perfusion.

[0110] Generally, this is adjusted by a process management system, such as the Lucullus PIMS, which, for example, uses a programmed step chain that itself reads pre-adjusted parameters or online signals from bioreactor probes to organize the perfusion process via a control pump. The addition of fresh culture medium is, for example, automatically triggered by the reduction in the weight of the bioreactor caused by the flow of the harvest and effluent leaving the bioreactor.

[0111] In a preferred embodiment, the method for perfusion cell culture according to the invention is run such that the volume of the cell suspension in the bioreactor (hereinafter also referred to as the liquid volume of the bioreactor) remains almost the same throughout the time of the method (excluding the filling and growth phases at the start of the cell culture). Almost the same or constant volume according to the invention means that the liquid volume of the bioreactor increases to no more than 110% of the initial volume, preferably no more than 105%, and decreases to no less than 90% of the initial volume, preferably no less than 95%.

[0112] In another preferred embodiment, the method for perfusion cell culture according to the invention includes continuous or semi-continuous perfusion and harvest. This means that throughout the time of the cell culture (excluding the filling and growth phases at the start), fresh cell culture medium is added to the bioreactor continuously or periodically, and the harvest is taken out of the bioreactor continuously or periodically. The amounts of perfusion and harvest can vary independently of each other. In one embodiment, the two liquid flows never completely stop.

[0113] In a highly preferred embodiment, in a system with a constant liquid volume in a bioreactor, and preferably also in a system with continuous or semi - continuous perfusion and harvest, each time an effluent is removed from the bioreactor, the harvest rate decreases due to the effluent rate such that the total volume of liquid removed from the bioreactor remains constant, varying by less than 10%, preferably less than 5%. In this way, the feed of fresh medium can be kept constant. This is preferred because in addition to the invariant harvest removal, removing the effluent would require adding an excessive amount of fresh medium to keep the bioreactor liquid volume constant or would result in a decrease in the liquid volume within the bioreactor. If the perfusion rate P and VCD are kept constant, then CSPR is also constant, which is a requirement for long - term steady - state operation.

[0114] In another preferred embodiment, if the liquid recovered from the effluent recovery device is pumped back into the bioreactor, the harvest rate increases. Preferably, it is increased to a rate such that the liquid volume of the bioreactor remains constant (varying by less than 10%, preferably less than 5%) without the need to modify the constant feed of fresh medium. This means that when the liquid recovered from the effluent recovery device is pumped back into the bioreactor, the harvest rate increases due to the rate of re - feeding of the liquid effluent.

[0115] Figure 4 Scheme showing exemplary flow rates of a perfusion method with 2 vvd according to the present invention. Each effluent recovery operation consists of 4 sequential stages, resulting in a characteristic viable cell density (VCD) curve (black line with black dots). In stage 1, the effluent pump (dashed line with light dots) is turned off and the harvest pump runs at 2 vvd (dotted line with black triangles) while the VCD is increasing. Stage 1 is the stage where no effluent operation is triggered. In stage 2, the effluent pump is set to 2 vvd (flowing into the effluent recovery device) while the harvest rate is stopped. At this time, the VCD decreases due to the removal of cells and the feed of fresh medium equal to the effluent rate. In stage 3, the on / off state of the pumps is equivalent to stage 1, except that the cells in the effluent recovery device settle. During this time, a biomass cake is accumulating. Stage 4 shows the re - feeding or effluent recovery operation: the effluent pump or the re - feeding pump runs at - 2 vvd (negative sign) because the direction of the pump is reversed (outflow of the effluent recovery device). The harvest pump runs at 4 vvd to compensate for the re - feeding of the effluent medium and maintain a constant feed of 2 vvd of fresh medium. During the operation of stage 4, the VCD increases only due to the remaining cells in the almost clarified re - feeding supernatant. When the biomass exceeds its set point, these sequential stages are re - triggered.

[0116] Such regulation of the liquid flow is preferably carried out by a process management system.

[0117] The present invention is also applicable to the CFB process.

[0118] The CFB process generally does not operate in a steady state because, due to the accumulation of products in the bioreactor, the loss of products through discharge would be extremely high. However, operating the CFB process in a bioreactor comprising an effluent recovery device according to the present invention and using the method according to the present invention also uniquely allows the CFB process to operate in a steady state because the effluent recovery minimizes the loss of products.

[0119] Without being limited thereto, the present invention is further illustrated by the following examples. All documents cited in the context and the corresponding European patent application EP 19207666.9 filed on November 7, 2019 are hereby incorporated by reference. Examples

[0120] Example 1: Evaluation of the design of the effluent recovery device by determining the sedimentation velocity of mammalian cells

[0121] Method:

[0122] A cylindrical glass column with an open top and a stopcock at the bottom was filled with a cell suspension (viable cell density: 30 x 10 6 vc / mL). The cylindrical column was vertically fixed in a tripod. The cells were then left in the column for a 6-hour period. Sedimentation was then monitored by reading the separation line between the cells and the fluid, which could be identified by the accumulation of cells below the line and the cell-free fluid above the line. This separation line moved downwards over time. The sedimentation length was read at specific time points. A graph was created with time [hours] on the x-axis and sedimentation length [cm] on the y-axis. The trend line was calculated and the slope or sedimentation velocity was obtained.

[0123] Results

[0124] Figure 6 The sedimentation trend line of the mammalian cell line over time was shown. The slope of the trend line was equal to the sedimentation velocity: 0.6889 cm / h.

[0125] Conclusion:

[0126] • The effluent recovery device can work best if it is designed as a flat shape with a short sedimentation length. The short sedimentation length makes it possible to reduce the separation time between the cells and the fluid.

[0127] • To handle the effluent volume of a perfusion bioreactor, the volume in the effluent recovery device must be distributed over a larger surface area.

[0128] • The device must be installed in a fixed manner without external shocks.

[0129] Example 2: Feasibility Test of the Design of a Flat Bag in Bioprocess Operations

[0130] Background Information:

[0131] The tests to determine the design are included within perfusion bioreactor operations. In such bioreactor runs, cells are inoculated at a low viable cell density of five hundred thousand cells / mL and expanded to 32 x 10 6 viable cells / mL. Then steady state is initiated and cells that would otherwise accumulate further through exponential growth are removed. In this example, the removed cells are pumped into a flat 2D effluent recovery bag rather than just in the waste. The effluent recovery operations in this experiment include tests of sedimentation time, proper handling of the pump, and testing of the bag design itself. The key questions to be answered through this experiment are as follows:

[0132] 1. How much time is required to sediment the cells as short as possible to obtain a stable biomass cake / clear supernatant

[0133] 2. On the premise of the previous question, can the clear supernatant be re-fed without increasing the biomass probe signal

[0134] 3. What is the possible pump speed before the biomass cake becomes unstable

[0135] 4. How much effluent can be recovered

[0136] Method:

[0137] To study the above questions, the hardware setup of the experiment was designed as follows:

[0138] An effluent pump (with adjustable speed and direction) is used in combination with a balance and a connected bioreactor. A trough with a 2D bag assembly is placed on the balance to measure the inflow and outflow.

[0139] A 2D plastic bag is placed in a white trough and horizontally fixed with tape. The designated inlet / outlet tubes are closed vertically with white clips leading to the pump. The weight of the trough is controlled by the balance and is used to quantify the liquid flow during the complete effluent recovery process.

[0140] The connection of the 2D plastic bag to the perfusion bioreactor is achieved with a specific tube assembly consisting of two sealable C-Flex tubes connected by a Pharmed pump tube:

[0141] The bioreactor is connected to the 2D bag via a pump through an effluent line pipe. Two sealable C-Flex tubes are installed for aseptic connection, and one tube is for the pump. An integrated sample port is used to control the inflow and outflow liquid flows for viable cell density and cell viability.

[0142] One of the sealable ends also contains a sample port for VCD control of the corresponding effluent suspension entering the effluent recovery device and / or the corresponding clarified supernatant returning to the bioreactor. The sample port is installed to answer questions 1.), 3.), and 4.).

[0143] Different from the common effluent pump directly installed on the bioreactor control unit, an independent pump with variable pump speed and direction is used. The answer to question 2.) will be derived from the reading of the biomass signal.

[0144] In this experiment, 3 runs of the hardware setup with the following process steps are carried out. The goal is to discharge half of the total daily discharge. Calculate half of the discharge based on the previous day's discharge:

[0145] 1. Discharge the calculated amount from the bioreactor at a maximum pump speed of 120 rpm (39.2 mL / min)

[0146] 2. Let the effluent settle for 120 minutes

[0147] 3. Re-feed the supernatant at different speeds / online control VCD biomass signal monitoring

[0148] Results:

[0149] Figure 7 Describe the sequential steps of the effluent recovery process: (1) Discharge the cell suspension from the bioreactor; (2) Sedimentation / separation of fluid and cell particles; (3) Re-feed operation of the clarified supernatant.

[0150] Figure 8 Describe the re-feed process (outflow) of the clarified supernatant in Run 1 with two different pump speeds. If the biomass cake is damaged by the pump, test with a lower pump speed of 2.29 mL / min. Even at 32 mL / min, the biomass cake remains stable. Compared with the discharge amount, the remaining share of the wet biomass is about 9.6%.

[0151] Figure 9 Describe the equivalent measurement of viable cell density using the sample port. After measuring an initial cell density of 3.3 x 10 6 vc / mL, it steadily decreases to below 0.5 x 10 6vc / mL. The initially high cell density comes from cell clusters adhering to the sample port tubing. The vertical increase in the curve indicates the point of biomass cake disruption or the maximum value of the supernatant re-fed to the bioreactor.

[0152] Overview of all runs:

[0153]

[0154] Conclusion:

[0155] The experiment of Example 2 was conducted to answer the following questions:

[0156] 1. How much time is required to sediment the cells as short as possible to obtain a stable biomass cake / clear supernatant

[0157] The setup of this experiment tested a sedimentation time of 120 minutes. During this period, a stable biomass cake was accumulated. The stability was demonstrated by the increase in pump speed and cell density monitoring. At an increasing pump speed from 2.29 to approximately 30 mL / min, it was maintained as long as the maximum fluid of the effluent was recovered.

[0158] 2. On the premise of the previous question, can the clear supernatant be re-fed without increasing the biomass probe signal

[0159] The relevance of the question is given because the biomass signal is the triggering influencing factor for the discharge process in a steady-state perfusion bioreactor. During the re-feed process, the biomass increases within the range of 2 - 4 pF. To evaluate the relevance of this increase, the normal fluctuations of the biomass signal during steady-state operation should be considered. Due to the alternating tangential flow filtration of the cell retention device or the feed / harvest process, the cell concentration and thus the biomass signal vary periodically over time. The observable range is also within the range observed during re-feed such as the effluent recovery operation. In the concept of this patent, the following conclusions can be drawn:

[0160] i. The biomass signal remains within the range that allows the perfusion process to further progress in a steady state

[0161] ii. The redundant effluent recovery device / process will be easy to compensate for a higher increase in the biomass signal that triggers the discharge operation.

[0162] 3. What is the possible pump speed before the biomass cake becomes unstable

[0163] In Run 1, an increase in pump speed from 2.29 to 32 mL / min was tested. The biomass cake there was not disrupted in any way.

[0164] 4. How much of the emissions can be recovered

[0165] The fraction of the remaining wet biomass cake is in the range of 9.47% - 14% compared to the emissions before the biomass cake was disrupted (indicated by the increase in cell density in the control sample).

[0166] These issues helped to further design the next experiment, in which a 2 - day steady - state operation for product recovery from emissions was tested.

[0167] Example 3: Emissions recovery operation in steady - state perfusion

[0168] Background information:

[0169] Example 2 answered the basic questions regarding technical feasibility. Example 3 describes complementary experiments showing the integration of the emissions recovery concept in a perfusion steady - state operation without operational interruptions. The question to be answered by this example is whether the biomass signal, which is the main parameter for steady - state control, terminates at a level similar to that before the start of the emissions recovery operation.

[0170] Method:

[0171] The perfusion bioreactor was run in perfusion with a mammalian cell line and an appropriate culture medium (perfusion rate = 3.66 vvd), first expanded and then transitioned to steady - state operation, with a biomass signal set - point of 86.5 pF (50 x 10 6 vc / mL). Based on the emissions of the previous day (0.66 vvd = 2772 g), the estimated value of the daily emissions was calculated and divided by 2 for two emissions recovery operations per day. When the emissions recovery operation started, the automated steady - state control of the biomass was turned off to manually control the steady - state in combination with the emissions recovery (see Figure 5 A).

[0172] Conceptually, the perfusion rate needs to remain constant throughout the emissions recovery operation. During the two emissions operations per day (rapidly removing half of the emissions per day), an equal amount of fresh culture medium was fed into the bioreactor, significantly increasing the perfusion during this short period. On the other hand, the perfusion rate decreased when there was no emissions operation. In this way, the total daily perfusion rate remained constant, although it varied during the day.

[0173] The experiment was divided into two phases ( Figure 5 B): After the emissions operation in the first phase, the cell suspension was directly re - fed back into the bioreactor, while in the second phase, a 60 - minute sedimentation time was applied between emissions and re - feeding. Both phases contained two emissions recovery operations.

[0174] Result:

[0175] In Figure 10 it is possible to see the first phase of the experiment:

[0176] Emission #1 / 2 at 31 rpm (= 640 mL / h; P = 3.66 vvd) within 130 minutes (emission 1386 g), no sedimentation: 3-step emission operation is carried out. Compared with the initial set point of 86.5 pF, the final biomass signal is 93.2 pF.

[0177] Between the first and second phases of Example 3, the biomass in the bioreactor is adjusted to 86.5 pF to have an equal starting point. In Figure 11 it is possible to see the second phase of the experiment.

[0178] Emission #1 / 2 at 31 rpm (= 640 mL / h; P = 3.66 vvd) within 130 minutes (emission 1386 g), 1-hour sedimentation: 3-step emission operation is carried out, and the final biomass signal is equal to the set point of 86.5 pF.

[0179] Conclusion:

[0180] In this example, the emission operation is integrated into the steady state of a real perfusion bioreactor. Two different sedimentation forms are tested to evaluate whether the biomass signal reaches the same value as before the emission recovery operation. Without sedimentation time, the refeeding of the emissions results in an increase in biomass in the bioreactor, while 60 minutes of sedimentation is suitable to end with the same biomass signal as before the emission recovery. Compared with Example 2 with a sedimentation time of 120 minutes, 60 minutes seems to be effective in Example 3. This can be explained by the longer emission time of 130 minutes in this setup (where cells may have started to sediment). Example 3 shows general applicability to the steady-state perfusion process. The calculation of the total harvest with and without emission recovery can be calculated using this example (Table 2):

[0181]

[0182] Table 2: Comparison of the harvest obtained between steady-state perfusion bioreactors with and without emission recovery operation. In addition to the usual harvest yield - an emission stream of 3.00 vvd, an emission of 0.66 vvd (without recovery operation) is further separated into a wet biomass cake of approximately 0.14 vvd that ultimately becomes waste and 0.52 vvd that is recovered and added to the harvest yield. The emission recovery operation in this example can increase the total harvest yield from 82% to 96%.

[0183] In Example 3, 17% more harvest can be collected compared to the standard steady-state method without emissions recovery.

Claims

1. A bioreactor system comprising a bioreactor having a perfusion inlet and a harvest outlet, wherein the bioreactor further comprises effluent recovery means having an inlet and an outlet for effluent, the inlet of the effluent recovery means directing the effluent from the bioreactor to means for separating the cells of the effluent from the liquid portion of the effluent, and the outlet of the effluent recovery means for directing the liquid portion of the effluent from the means for separating the cells of the effluent from the liquid portion of the effluent to the bioreactor, wherein the liquid portion of the effluent comprises liquid cell culture medium, waste and target product.

2. The bioreactor system according to claim 1, comprising at least one pump for controlling the flow of the effluent into the effluent recovery means and for controlling the flow of the liquid portion of the effluent out of the means for separating the cells of the effluent from the liquid portion of the effluent through the outlet of the effluent recovery means back into the bioreactor.

3. The bioreactor system according to claim 2, wherein the at least one pump is for controlling the flow of the effluent into the means for separating the cells of the effluent from the liquid portion of the effluent.

4. The bioreactor system according to claim 1 or 2, wherein the system further comprises a process management system.

5. The bioreactor system according to claim 1 or 2, wherein the inlet and outlet of the effluent recovery means comprise plastic tubing.

6. The bioreactor system according to claim 1 or 2, wherein the outlet of the effluent recovery means directs into the bioreactor.

7. The bioreactor system according to claim 1 or 2, wherein the means for separating the cells of the effluent from the liquid portion of the effluent is a container for sedimenting the cells.

8. The bioreactor system according to claim 1 or 2, wherein the outlet of the effluent recovery means is on the opposite side of and connected to the container for sedimenting the cells at the upper half of the opposite side.

9. The bioreactor system according to claim 1 or 2, wherein the means for separating the cells of the effluent from the liquid portion of the effluent is a container for sedimenting the cells, which is a flat container, the minimum dimension of which is its height when placed horizontally.

10. The bioreactor system according to claim 9, wherein the flat container is a plastic bag.

11. A method for perfusing cell culture, comprising culturing cells in a bioreactor system comprising a bioreactor having a perfusion inlet and a harvest outlet and effluent recovery means having an inlet and an outlet for effluent, the inlet of the effluent recovery means for directing the effluent from the bioreactor to means for separating the cells of the effluent from the liquid portion of the effluent, and the outlet of the effluent recovery means for directing the liquid portion of the effluent from the means for separating the cells of the effluent from the liquid portion of the effluent back into the bioreactor, wherein i. Continuously or one or more times during the cell culture process, insert fresh cell culture medium into the bioreactor through the perfusion inlet; ii. Continuously or one or more times during the cell culture process, withdraw the harvest from the bioreactor through the harvest outlet; and iii. Continuously or one or more times during the cell culture process, withdraw a certain amount of the effluent from the bioreactor through the inlet of the effluent recovery device and direct it to a device for separating the cells of the effluent from the liquid portion of the effluent, separate the cells and the liquid portion, and transfer the liquid portion back to the bioreactor through the outlet of the effluent recovery device, wherein the liquid portion of the effluent contains liquid cell culture medium, waste, and the target product.

12. The method according to claim 11, wherein in iii, the liquid portion of the effluent transferred back to the bioreactor contains more than 70% of the liquid contained in the effluent withdrawn from the bioreactor and less than 5% of the cells contained in the effluent withdrawn from the bioreactor.

13. The method according to claim 11 or 12, wherein iii is carried out one or more times during the cell culture process, a certain amount of the effluent is withdrawn from the bioreactor through the inlet of the effluent recovery device and directed to a device for separating the cells of the effluent from the liquid portion of the effluent, the device comprising a container that allows the effluent to settle in the container for a period of time such that the cells settle towards the bottom surface of the container, and then the cell-depleted liquid supernatant above the settled cells is withdrawn through the outlet of the effluent recovery device and returned to the bioreactor.

14. The method according to claim 13, wherein the time allowed for the effluent to settle in the container is between 15 minutes and 2 hours.

15. The method according to claim 11 or 12, wherein the method steps i, ii, and iii are adjusted such that the volume of the cell culture in the bioreactor remains at a constant level.

16. The method according to claim 11 or 12, wherein the method is a CFB method in a bioreactor system including an ultrafiltration membrane.

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